Effects of aging and exercise training on the histological and mechanical properties of articular...

Biogerontology (2012) 13:369–381 DOI 10.1007/s10522-012-9381-8

RESEARCH ARTICLE

Effects of aging and exercise training on the histological and mechanical properties of articular structures in knee joints of male rat Hideki Moriyama • Naohiko Kanemura • Inge Brouns • Isabel Pintelon • Dirk Adriaensen • Jean-Pierre Timmermans • Junya Ozawa • Nobuhiro Kito • Toshiaki Gomi • Masataka Deie

Received: 13 December 2011 / Accepted: 12 April 2012 / Published online: 22 April 2012 Ó Springer Science+Business Media B.V. 2012

Abstract The impact of aging on joints can have a profound effect on an individual’s functioning. Our objectives were to assess the histological and mechanical properties of the knee joint capsule and articular cartilage with aging, and to examine the effects of exercise on age-related changes in the knee joint.

2-year-old Wistar rats were divided into a sedentary control group and an exercise-trained group. 10-weekold animals were used to investigate the changes with

Hideki Moriyama and Naohiko Kanemura contributed equally to this work.

H. Moriyama (&) M. Deie Graduate School of Health Sciences, Hiroshima University, Hiroshima 734-8551, Japan e-mail: morihide@harbor.kobe-u.ac.jp Present Address: H. Moriyama Graduate School of Health Sciences, Kobe University, Tomogaoka 7-10-2, Suma-ku, Kobe 654-0142, Japan N. Kanemura T. Gomi School of Health and Social Services, Saitama Prefectural University, Saitama 343-8540, Japan I. Brouns I. Pintelon D. Adriaensen J.-P. Timmermans Laboratory of Cell Biology and Histology, University of Antwerp, 2020 Antwerp, Belgium J. Ozawa N. Kito Department of Physical Therapy, Hiroshima International University, Hiroshima 739-2695, Japan

aging. The joint capsule and cartilage were evaluated with histological, histomorphometric, immunohistochemical, and mechanical analyses. Severe degenerative changes in articular cartilage were observed with aging, whereas exercise apparently did not have a significant effect. The articular cartilage of aged rats was characterized by damage to the cartilage surface, cell clustering, and an abnormal cartilage matrix.

Histomorphometric analysis further revealed changes in cartilage thickness as well as a decreased number of chondrocytes. Aging led to stiffness of the articular cartilage and reduced the ability to dissipate the load and distribute the strain generated within the joint.

Joint stiffness with aging was independent of capsular stiffness and synovitis was not a characteristic feature of the aging joint. This study confirms that aging alone eventually leads to joint degeneration in a rat model.

The lack of recovery in aging joint changes may be due to several factors, such as the duration of the intervention and the regeneration ability of the cartilage.

Keywords Aging Joint capsule Cartilage Exercise Rat

Introduction Aging joint and muscle changes can have a tremendous impact on an individual’s overall functioning (Ahmed et al. 2005). Although the morphological, metabolic, and contractile properties of aging muscle

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have been extensively studied in rat models, much less information is available on age-related changes in the lower extremity joints. While the thickness of tibial cartilage did not substantially decrease in 26-monthold rats, its histological structure was changed when compared to young adult rats (Gyarmati et al. 1987).

Similar results were found in 24-month-old Wistar rats, 68 % of which displayed minimal to mild lesions in the medial tibial plateau that were characterized by loss of proteoglycan staining in the superficial zone accompanied by loss of chondrocytes (Smale et al.

1995). Furthermore, the thickness and cellular density of articular cartilage in the femoral trochlea was found to be diminished in 32-month-old rats (Oda et al.

2007) and elevated chondrocyte apoptosis rates were found in the calcified layer of knee cartilage in 24-month-old rats (Adams and Horton 1998). In the lower limb joints, the articular surface of the femoral heads in 21-month-old rats was characterized by empty lacunae of chondrocytes and numerous exposed collagen fibrils (Gattone et al. 1982). These studies have been focused on the age-dependent alterations in the articular cartilage and on only one of morphological changes taking place in the main components of the cartilage matrix. Osteoarthritis (OA) increases in prevalence with aging and is a degenerative joint disease generally characterized by progressive cartilage degeneration (Martin and Buckwalter 2002); therefore, these studies have been described in association with OA. Joints are composed of several different tissues (cartilage, capsule, synovium, and ligament) that interact in unknown ways to allow joints to function relatively well (Burr 2004). These tissues are all important to the optimal functioning of joints, and when one tissue begins to deteriorate, it inevitably affects on the others (Burr 2004). Taken together, earlier experiments in only part of the cartilage are insufficient to understand the changes in the joint with advancing age.

Joint stiffness is frequently observed in elderly people (Trichard et al. 1982) and individuals with knee OA (Dixon et al. 2010), and increases the joint’s vulnerability to injury (Ahmed et al. 2005). The joint capsule is known to be of vital importance to the function of a synovial joint. It forms part of the seal that keeps lubricating synovial fluid in position, provides passive stability by limiting joint movements, and active stability via its proprioceptive nerve endings (Ralphs and Benjamin 1994). Earlier studies emphasized the

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pathogenetic role of muscles causing joint stiffness (Ochi et al. 2008), whereas tissues within the articular structures have been poorly documented. We hypothesize that joint stiffness results from capsular stiffness.

It is widely accepted that appropriate exercise counteracts the progressive loss of muscle mass, muscle strength, and quality (Larsson and Ramamurthy 2000). Even short-term (1 month) treadmill exercise training started at an advanced age was able to reverse age-related skeletal muscle apoptosis and thus proved to be effective strategy for improving physical performance and muscle strength in old rats (Marzetti et al. 2008). On the other hand, the effects of exercise training on the age-related changes in rat joints have not been studied experimentally. Animal studies in healthy young joints have shown that short-term moderate or strenuous exercise does not cause cartilage degeneration and does not have a deleterious effect on the mechanical properties of canine cartilage (Newton et al. 1997). Additionally, animal studies have shown that disruption of proprioception in a joint leads to advanced and accelerated degenerative changes of the joint (O’Connor and Brandt 1993). Studies comparing knee proprioception in active elderly and sedentary elderly participants suggested that regular exercise attenuated the decline in proprioceptive seen with aging (Petrella et al. 1997). Exercise intervention also has been reported to influence joint stiffness to varying effects depending on age (Ochi et al. 2008).

The main aims of the present study were to investigate the histological and biomechanical changes occurring in the knee joint capsule and articular cartilage with (physiological) aging, and to examine the effects of exercise on these age-related changes. Biomechanical studies are essential since structural and biochemical changes have been associated with alterations in the mechanical properties of the tissue (Akizuki et al. 1986).

The functional biomechanical properties of a tissue, therefore, best reflect the complexity of fundamental structural changes (Poole et al. 2010).

Materials and methods Experimental design The protocols for the experiments were approved by the Committee of Research Facilities for Laboratory Animal Science of Hiroshima University School of

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Medicine. Animal model systems represent an important surrogate for studies of aging in humans (Aigner et al. 2010). Given the high cost of maintaining larger species, smaller animal models are preferred for preliminary screening, and rat models are frequently used for initial assessment of in vivo efficacy. The most efficacious compound in a rat model may subsequently be tested in a larger animal model prior to setting up human trials (Gerwin et al. 2010). In the present study, 10 old male Wistar rats (CLEA Japan Inc., Tokyo, Japan; age: 24 months; weight 400–520 g) were used and divided into a sedentary control group and an exercise group. The half-mortality age of Wistar rats is 24 months and the maximal lifespan is 36 months (Larsson and Ansved 1995). Animals of 24 months of age were used to avoid the unpredictable effects of disease processes. Four animals were used as sedentary controls. The remaining six rats were trained on a treadmill or a custom balance exercise device for 1 month (5 times a week). Three rats were introduced daily to the treadmill walking at a speed of 11.8 m/min for 1 h. Walking speed and the duration of exercise were calculated so as to reflect a work rate of *40 % maximal O2 uptake in the animals (Lawler et al. 1993).

Three animals were placed on a moving platform (angle of inclination, ±7°; number of revolutions, 25 rpm) for 1 h a day to attenuate the decline in proprioception seen with aging (Ahmed et al. 2005; Petrella et al. 1997). The balance exercise program was intended to recruit a combination of isometric, concentric, and eccentric activities of the lower-extremity musculature (Brown and Taylor 2005). In addition, four adolescent male Wistar rats (age: 10 weeks; weight: 172–186 g) were used as a young group with healthy joints to investigate age-related changes. All animals were housed in rooms with a temperature set at 23 ± 1 °C, relative humidity between 50 and 60 %, and a 12/12 h light/dark cycle. Only active rats with proper food intake and without evident motor deficits or visible pathological signs were admitted to the study.

Histological evaluation At the end of the maintenance intervals, the animals were killed by exsanguination under anesthesia. The femur and tibia were dissected free of soft tissues and disarticulated at the hip and ankle. The knee joints spontaneously assumed an extended position at 125°

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when the restraint of surrounding soft tissues was removed (O’Connor 1997). We prepared undecalcified frozen sections following the protocols established by Kawamoto (2003). The unilateral knee joints including the patella and joint capsule were resected and immediately frozen in a mixture of hexane and isopentane cooled with a cooling apparatus (UT2000F, FINETEC, Tokyo, Japan). Each sample was embedded in 5 % carboxymethyl cellulose gel and completely frozen. Each undecalcified frozen block was then attached to the sample stage of a cryomicrotome (CM3050S, Leica Microsystems, Tokyo, Japan) in a cryochamber. The block was covered with a polyvinylidene chloride film (CF0114, Leica Microsystems) and sectioned with the film at a thickness of 5 lm using a disposable tungsten carbide blade (Jung TC-65, Leica Microsystems). The knees were aligned so that sagittal plane sections were cut in the proximal-to-distal direction at the medial and lateral mid-condylar level in the sagittal plane. These sections were stained with aldehyde fuchsin–Masson Goldner for the joint capsule, and toluidine blue or safranin-O/fast green for the articular cartilage.

Quantitative histology Determination of measurement sites We quantified the histological changes in articular cartilage as described previously (Moriyama et al.

2008, 2009). Cartilage alterations after immobilization (Hagiwara et al. 2009; Trudel et al. 2005) or unloading (O’Connor 1997) may differ between different cartilage plates of the knee, based on the different mechanical conditions specific to each site. Therefore, changes in femoral and tibial cartilage at the medial and lateral mid-condylar level were determined in the 12 regions by slight modification of the method applied in our previous study (Moriyama et al. 2008, 2009). Cartilage regions were defined according to their positions in embedded joints where the knee joint was positioned at an angle of 125°. The anterior femoral (FA) and middle tibial (TM) regions were defined as the regions of articular cartilage located between the inner edges of the anterior and posterior meniscal horns. The edge of the posterior femoral (FP) region was located 20 lm beyond the outer edge of the posterior meniscal horn, and the middle femoral (FM) cartilage region was situated between the FA and FP regions. The anterior

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tibial (TA) region was located adjacent to the anterior horn of the meniscus, and the posterior tibial (TP) region was adjacent to the posterior horn.

Cartilage thickness Articular cartilage thickness was measured on sections stained with toluidine blue, which provides excellent color discrimination between bone and calcified cartilage, as well as a distinct basophilic line that marks the location of the tidemark (O’Connor 1997).

Cartilage thickness was defined as the distance between the cartilage surface and the osteochondral junction. Because of the undulating nature of the osteochondral junction, and the consequent large sitedependent variability in cartilage thickness, we measured an area of cartilage and calculated the mean thickness over that area (Trudel et al. 2005). Histological sections were digitized by a 49 microscope objective with a light microscope (BX51, Olympus, Tokyo, Japan) and a camera (DP70, Olympus). In each region, a 400-lm-long stretch of the cartilage surface was defined and the cartilage area under this stretch was measured, following the osteochondral junction.

The mean thickness of the cartilage was calculated by dividing the area by 400 lm.

Number of cells Histological sections stained with safranin-O/fast green were digitized by a 209 microscope objective with a light microscope and a camera. Rectangles 100 lm deep and 400 lm long were superimposed over histologic sections in each of the 12 regions.

Chondrocytes were manually counted within the rectangular field.

Immunohistochemical analysis We assessed age- and exercise-related changes in immunohistological staining patterns at the joint capsule and in the articular cartilage. Frozen sections were air-dried, fixed in ethanol for 2 min, and rehydrated in 0.01 M phosphate-buffered saline (PBS, pH 7.4) for 5 min. Sections were treated with 0.5 % bovine testicular hyaluronidase (H3506, SigmaAldrich Co., MO, USA) in PBS for 60 min at room temperature. After two rinses with PBS for 5 min

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each, endogenous peroxidase was inactivated by incubation of the sections in methanol containing 0.3 % H2O2 for 20 min. Non-specific binding sites were blocked by treating the sections with 1 % normal serum from the host species of the secondary antibody (horse or goat) in PBS. After removal of the blocking solution, sections were incubated with mouse monoclonal anti-collagen type I (diluted 1:4,000; C2456, Sigma-Aldrich Co.), anti-collagen type III (diluted 1:8,000; C7805, Sigma-Aldrich Co.), antiCD31 (diluted 1:250; MCA1334GA, AbD Serotec, Oxford, UK), or anti-pentosidine (diluted 1:50; KH012, Trams Genic Inc., Kumamoto, Japan) antibodies for the joint capsule, and mouse monoclonal anti-collagen type II (diluted 1:1,000; F-57, Daiichi Fine Chemical Co., Toyama, Japan) or goat polyclonal anti-matrix metalloproteinase 13 (MMP-13; diluted 1:2,000; AB8120, Millipore Co., Billerica, MA, USA) antibodies for the articular cartilage overnight at 4 °C. Sections were then rinsed in PBS and reacted with horse biotinylated anti-mouse IgG (diluted 1:250; BA2001, Vector Laboratories, Burlingame, CA, USA) or rabbit biotinylated anti-goat IgG (diluted 1:30; Histofine 416022, Nichirei Biosciences Inc., Tokyo, Japan) for 1 h at room temperature. A subsequent reaction was made by the streptavidin–biotin-peroxidase complex technique using an Elite ABC kit (diluted 1:50; Vector Laboratories) for 30 min. Immunoreactivity was visualized with 3,30 -diaminobenzidine tetrahydrochloride (K3466, Dako Japan, Tokyo, Japan), followed by counterstaining with methylene green.

The primary antibody was omitted for the negative controls.

Mechanical analysis Each specimen assigned to the mechanical analysis of the contralateral knee joint was stored at -80 °C until mechanical tests. We performed two series of mechanical tests as described by published studies (Akai et al.

1993; Usuba et al. 2007). These tests were nondestructive, dynamic forced vibration methods to examine viscoelasticity (phase lag and dynamic stiffness), and a static destruction test with tensile force. The specimens were thawed at room temperature just before mechanical analysis and were kept moist with normal saline at all times. Testing was completed within 4 h of thawing.

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Mechanical testing of the viscoelasticity of the articular structure The first mechanical test evaluated the viscoelasticity of the joint capsule and the articular cartilage by spectral analysis based on the fast fourier transform of displacement. A spectrum analyzer (3582A, Hewlett Packard, Palo Alto, CA, USA) served as a dualchannel fast fourier transform machine and provided electrical driving signals to the viscoelastic spectrometer (DDV-VMF, Orientec Co., Tokyo, Japan). A small rod, which was connected to the load cell of the spectrometer, applied the forced vibration at a frequency of 1 Hz to the distal end of the tibia and 10 Hz to the cartilage surface (weight-bearing area) of the femur or tibia at the medial and lateral mid-condylar level. The transfer functions, the mathematic relation between both input and output signals through the specimen, indicated a delay of phase (phase lag) and structural stiffness (dynamic stiffness). Phase lag (tan d) indicated the shock-absorbing ability and dynamic stiffness (N/mm) referred to the transform resistance at the primary phase of lengthening.

Static destruction testing of the periarticular connective tissue Another mechanical test, the tensile test of the bonejoint-bone complex, was determined from the loaddeformation curves. A load cell, which was attached to a traction device (Tensilon UTM-10T, Orientec Co.) that moved to hyperextend the joint at 3 mm/min, was applied to the distal end of the tibia. The load and deformation of each joint were continuously recorded with a xy-recorder. The liner slope of the load-deformation curve was used to calculate peak maximum load (N) and stiffness (N/mm) of the joint.

Statistical analysis The software program JMP 7 (SAS Institute, Cary, NC, USA) was used for the statistical analysis.

Descriptive statistics were calculated as median and interquartile range (IQR). An alpha level of 0.05 was used for all statistical tests, and two-tailed tests were applied. The Kruskall–Wallis nonparametric test was used to evaluate the differences among the groups which were not normally distributed (Zar 2010). When statistical significance was achieved, a post hoc Welch

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test was used to further specify the difference between the groups. For the post hoc analysis, the Shaffer correction was applied to adjust the a priori alpha level to the number of comparisons performed.

Results Given the lack of differences between the treadmill and balance exercise groups, the results for all outcome measures were pooled into one comparison (exercise-trained) group for proper comparison of the effects of aging on all outcome measures.

Histological findings Microscopic examination showed severe degenerative changes in the articular cartilage with aging, although no difference was observed between the sedentary and exercise-trained groups in any region. Histological findings revealed fissures, enlarged lacunae without nuclei corresponding to chondrons, and clustering of chondrocytes, particularly in the medial tibial cartilage (Fig. 1a–c). On the other hand, evidence of inflammation, such as the presence of inflammatory cells (e.g., polynucleated cells, lymphocytes, macrophages), and proliferation of connective tissue was not observed at the joint capsule. No apparent degeneration of the joint capsule or articular cartilage was found in any of the specimens from young rats.

Total cartilage thickness (including both uncalcified and calcified layers) did not differ among the groups in any region of the femur or tibia at the medial or lateral levels (P [ 0.10). At the medial midcondylar level of the knee, only the uncalcified layer in the FA cartilage region was thinner in the sedentary (P = 0.046) and exercise-trained (P = 0.016) aged groups than in the young group. Accordingly, at the lateral mid-condylar level, the uncalcified layer in both the FA (P = 0.0002) and FP (P = 0.006) cartilage regions was thinner in exercise-trained old rats than in young rats, although cartilage thickness in sedentary rats was comparable with that in young rats.

The calcified layers in the FA, FM, FP, and TP cartilage regions were thicker in the sedentary and exercise-trained groups than in the young group (P \ 0.038) at medial level. The lateral level, unlike the medial level, did not show any significant differences in femoral or tibial calcified cartilage among the

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b Fig. 1 Typical histopathological changes in rat knees. Evi-

dence of fissures on the cartilage surface and cluster formation are obvious in an old rat (compared with the young rats) (a–c).

Aging induced advancement of the tidemark (arrowheads) toward the joint surface, resulting in a thinning of the uncalcified cartilage layer and a concurrent thickening of the calcified layer (d–f). a–c: Medial TM cartilage; Safranin-O and light green staining. d–f: Lateral TP cartilage; Toluidine blue staining.

Immunohistochemical detection of the distribution of collagen type II in the lateral TA cartilage (g–i), MMP-13 in the medial TP cartilage (j–l), and CD31 in the lateral synovial membrane between the patellar tendon and femur (m–o). Collagen type II was present in large quantities over all layers in young cartilage (g) but decreased with aging (h, i). Cartilage in the young group solely expressed MMP-13 (j), whereas the pericellular regions in the old rat groups were clearly stained (arrows) (k, l).

Immunostaining for CD31 showed intense labeling in the young group (m) but weak labeling in the old groups (n, o). Scale bars 100 lm (a–c, j–o) and 200 lm (d–i). AC articular cartilage, SB subchondral bone

groups (P [ 0.05). The mean uncalcified layers expressed as a percentage of total cartilage thickness are shown in Table 1. The tidemark advanced toward the joint surface with aging, leading to a shift in the proportion of uncalcified versus calcified cartilage (Fig. 1d–f). As compared with the young group, the proportion of uncalcified cartilage was significantly smaller in the sedentary and exercise-trained groups in the FA, FM, FP, and TP cartilage regions (all P \ 0.05).

Table 2 shows the number of chondrocytes in femoral and tibial cartilage. The number of chondrocytes differed among the various cartilage plates of the knee. At the medial level, there were significantly fewer chondrocytes in the sedentary and exercise-trained old knee joints than in young knee joints in FA, FP, and TP cartilage (P \ 0.029). In contrast, no statistically significant differences were found at the lateral cartilage level among the different groups (P [ 0.05).

Immunohistochemical observations Only weak immunolabeling for collagen type II was observed over all layers of the articular cartilage with aging at both the medial and lateral levels, whereas collagen type II was abundantly expressed in young cartilage (Fig. 1g–i). MMP-13 immunostaining showed intense expression in pericellular regions from the middle to deep cartilage layers in both the sedentary and exercise-trained groups (Fig. 1j–l). Staining intensity and the expression pattern of collagen types I and III did not appear to differ among the groups in any joint capsule (data not shown). Accordingly, pentosidine labeling showed no difference among the groups either. CD31-immunoreactive blood vessels were predominantly distributed throughout the synovial membrane of young knees, while only weak reactivity

Table 1 Mean uncalcified layer expressed as a percentage of whole cartilage thickness Regions

Young

Sedentary old

Exercised old

P values

Power

FA

90.13 (86.22, 93.43)

61.75 (55.88, 66.12)*

62.21 (51.16, 64.28)*

0.015

0.996

FM

89.15 (88.29, 90.21)

68.77 (60.19, 69.36)*

62.24 (61.79, 64.33)*

0.017

0.936

FP

93.15 (91.97, 94.98)

62.18 (57.64, 65.65)*

59.46 (44.80, 75.83)*

0.018

0.878

TA

69.50 (63.43, 75.14)

52.22 (50.98, 55.66)

67.57 (43.11, 76.41)

0.304

0.148

TM

75.84 (64.93, 86.29)

55.62 (45.62, 65.46)

55.42 (49.94, 65.21)

0.291

0.277

TP Lateral

87.54 (81.75, 88.91)

48.29 (41.90, 53.90)*

55.93 (54.84, 58.36)*

0.012

0.889

FA

94.09 (87.48, 96.24)

64.75 (56.15, 74.29)

53.70 (43.24, 72.97)*

0.047

0.689

FM

82.59 (77.70, 86.56)

58.56 (47.81, 69.30)

61.25 (57.59, 72.29)

0.075

0.480

FP

89.24 (82.67, 93.93)

58.50 (48.32, 67.05)*

54.86 (42.96, 60.05)*

0.039

0.830

TA

73.07 (70.05, 77.40)

65.59 (49.58, 77.12)

66.83 (57.90, 74.35)

0.695

0.142

TM

83.01 (79.75, 87.73)

47.12 (30.69, 68.02)

66.13 (63.26, 72.99)

0.045

0.616

TP

85.59 (83.11, 89.88)

54.27 (45.22, 61.79)*

50.11 (45.22, 60.28)*

0.018

0.983

Medial

Displacement values are given as median (IQR) % P value by Kruskal–Wallis H statistic * Significantly different from young group

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Table 2 Number of chondrocytes in a 100 lm by 400 lm area of articular cartilage Regions

Young

Sedentary old

Exercised old

P values

Power

Medial FA

74.00 (68.25, 75.75)

34.50 (23.50, 48.75)*

35.00 (23.50, 39.00)*

0.032

0.869

FM FP

80.50 (68.75, 86.50) 105.50 (91.00, 112.50)

42.00 (30.50, 51.00) 36.50 (27.75, 50.00)*

52.00 (42.00, 58.50) 42.00 (40.50, 49.00)*

0.068 0.029

0.689 0.974

TA

106.00 (98.75, 115.50)

78.00 (66.25, 86.00)

60.00 (56.00, 74.50)

0.034

0.759

TM

122.00 (101.25, 130.00)

64.00 (37.00, 88.50)

47.00 (33.00, 60.50)

0.055

0.880

TP

102.50 (92.50, 108.50)

41.00 (33.00, 53.75)*

51.00 (50.50, 62.00)*

0.018

0.964

FA

94.50 (78.75, 107.00)

51.50 (35.25, 65.50)

59.00 (40.00, 84.50)

0.114

0.446

FM

98.00 (89.00, 108.25)

53.50 (36.25, 71.75)

65.00 (57.00, 71.50)

0.084

0.731

FP

97.50 (75.00, 120.75)

67.50 (45.50, 84.75)

62.00 (51.00, 86.50)

0.321

0.253

TA

107.00 (101.25, 118.00)

70.00 (43.50, 96.25)

69.00 (65.00, 92.50)

0.056

0.464

TM

96.00 (84.25, 108.25)

64.50 (39.50, 86.50)

71.00 (57.50, 89.00)

0.214

0.303

TP

80.00 (68.00, 100.50)

69.50 (59.00, 76.25)

68.00 (61.50, 78.50)

0.416

0.284

Lateral

Displacement values are given as median (IQR) P value by Kruskal–Wallis H statistic * Significantly different from young group

was found in the old knees (Fig. 1m–o). Exercise had no significant effect on these staining patterns.

Mechanical properties Viscoelasticity (phase lag and dynamic stiffness) and static destruction (peak maximum load and stiffness) of sedentary old and exercise-trained old knees were comparable to those of young knees (P = 0.180, power = 0.431; P = 0.374, power = 0.133; P = 0.873, power = 0.085; P = 0.341, power = 0.272; respectively) in periarticular connective tissue (data not shown).

The viscoelasticity of articular cartilage significantly differed among the groups (P \ 0.05). Although no significant effect of exercise was detected in articular cartilage (P [ 0.05), dynamic stiffness was greater in old rats at the medial femur (P = 0.013, power = 0.999) (Fig. 2a) and phase lag was lower at the medial (P = 0.022, power = 0.884) (Fig. 2b) and lateral tibia (P = 0.013, power = 0.967) (Fig. 2c).

Discussion In this study, we characterized the natural history of the joint capsule and cartilage in rat knee joints, and examined the effects of exercise training on age-related

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changes to joints. Aged animals had a high incidence of cartilage lesions, although no effect of exercise was detected.

We found a percentage reduction in thickness of the uncalcified layer as well as advancement of the tidemark with aging. Tidemark advancement may accelerate after the age of 60 in humans (Lane and Bullough 1980) and it has been suggested that the tidemark may be reactivated by trauma, joint unweighting, or during OA, leading to a thinning of the uncalcified layer of cartilage (Bullough 1981; O’Connor 1997; Radin et al. 1991). Articular cartilage calcification is a well-known phenomenon observed in late-stage OA (Ea et al. 2011). The results of the present study are at least partially consistent with these earlier findings. In a non-diseased joint, although tidemark advancement makes the calcified cartilage thicker, remodeling at the osteochondral junction occurs more quickly, which causes the calcified cartilage to become thinner (Burr 2004). With aging, both tidemark advancement and subchondral remodeling accelerate resulting in a thicker calcified cartilage layer (Burr 2004). This process is accompanied by a reduction in the thickness of uncalcified cartilage. Mechanical stresses in the articular cartilage are likely to increase as this process progresses. Under normal conditions, articular cartilage and subchondral bone act together in transmitting load pressure through joints; therefore, the

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A

P = 0.001

160

Dynamic stiffness (N/mm)

377

P = 0.019

140 120 100 80 60 40 20 0 Young

Sedentary old

Exercised old

B 0.20 P = 0.021

0.18

P = 0.020

Phase lag (tan δ)

0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 Young

Sedentary old

Exercised old

C P = 0.004

0.20 P = 0.012

0.18

Phase lag (tan δ)

0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 Young

Sedentary old

Exercised old

Fig. 2 Viscoelastic analysis (phase lag and dynamic stiffness) in each group. Dynamic stiffness of medial femoral cartilage (a).

Phase lag of medial tibial cartilage (b). Phase lag of lateral tibial cartilage (c). The horizontal bars indicate the median and the vertical bars the range. The horizontal boundaries of the boxes represent the first and third quartiles

integrity of both tissues is required for adequate function (Bobinac et al. 2003; Eckstein et al. 1992, 1998). This study demonstrated that aging leads to stiffening of articular cartilage (medial femoral condyle) and reduces the ability to dissipate the load and distribute the strain

generated within the joint (tibial plateaus). From a mechanical perspective, this would increase mechanical stresses, thereby increasing damage to articular cartilage.

Aging is associated with progressively reduced cellularity in articular cartilage (Barbero et al. 2004; Oda et al. 2007; Temple et al. 2007), probably as a consequence of cell death over time (Adams and Horton 1998; Grogan and D’Lima 2010). The ability of chondrocytes to proliferate, and hence also to repair and maintain the cartilage matrix seems to decrease with age (Ahmed et al. 2005). Furthermore, the multifaceted nature of joint pathologies suggests that the contribution of cell death and cluster formation is an important factor in early- and late-stages OA (Grogan and D’Lima 2010). In addition to earlier reported age-related changes in articular cartilage (Adams and Horton 1998; Gyarmati et al. 1987; Smale et al. 1995), we also confirm marked differences between the medial and lateral knee joint. Changes in mechanical loading of chondrocytes caused by damage to and loss of matrix molecules contribute to the degenerative pathology, because excessive and noncyclic loading can stimulate cartilage degeneration in vitro (Poole et al. 2010). Therefore, the decline in cell numbers observed in the medial region only may be explained by articular surface incongruity resulting from changes in cartilage thickness.

Collagen type II, the main collagen type of hyaline cartilage responsible for the stability and cell biological functions of healthy articular cartilage (Poole 1999; Prockop et al. 1979), originates from chondrocytes (Doherty et al. 1998; Hagiwara et al. 2010a).

MMP-13 is capable of degrading type II collagen at a much higher rate than other collagenases (Bramono et al. 2004), and its expression is highly increased in articular cartilage in response to joint injury (Hayami et al. 2004). We observed collagen damage at the articular surface extending deeper into the cartilage in the aging joint as well as increased immunostaining of MMP-13 at the pericellular regions from the middle to deep cartilage layers, which was not seen in younger cartilage. Damage to the collagen type II meshwork is a critical event in the pathology of OA (Hagiwara et al.

2010a; Henrotin et al. 2007) and increased expression of MMP-13 in human OA cartilage and in OA animal models has been well documented (Ando et al. 2009).

Determining the levels of structural collagens is a vital element in understanding the elastic changes

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taking place in the capsule (Hagiwara et al. 2010b; Moriyama et al. 2007). The major structural collagens of the capsule are collagen types I and III, with the former accounting for 83 % of all collagen present (Hagiwara et al. 2010b; Kleftogiannis et al. 1994).

Collagen type I is found in tissues requiring high levels of mechanical strength (Hayashi and Nagai 1981) and its expression is increased at sites where new fibrosis and connective tissue proliferation occur (Matsumoto et al. 2002; Schollmeier et al. 1996). Increased collagen cross-linking is central to stiffening of the collagen network and loss of flexibility in articular structures (Ahmed et al. 2005). Earlier studies have reported that the concentration of pentosidine, one of the key proteins associated with joint stiffness, increased with age, giving rise to increased collagen cross-linking (Bank et al. 1998). Unexpectedly, the results of this study suggest that accumulation of collagen type I and pentosidine did not cause joint stiffness and that changes in the capsule did not result from proliferative fibrosis. The mechanical properties of the capsule remained unchanged with aging, which further indicates that joint stiffness with aging is independent of capsular stiffness. Joint stiffness probably results from a combination of age-related changes seen in articular structures (e.g., muscles and articular cartilage) other than the joint capsule.

Collagen type III is abundant in tissues that require high levels of mechanical compliance (Bornstein and Sage 1980; Hayashi and Nagai 1981). It is a major constituent of normal synovial membranes (Bland and Ashhurst 1997) and is also present in the inflamed and rheumatoid synovial membrane (Adam et al. 1976; Hagiwara et al. 2010b; Weiss et al. 1975). Synovial angiogenesis has been linked to histological synovitis, in particular macrophage infiltration of the synovium (Haywood et al. 2003; Walsh et al. 2007), and is stimulated during the inflammatory response that accompanies the pathological progression of OA (Appleton et al. 2007; Im et al. 2010). Blood vessel formation has been found to be substantially increased in the synovium in knee joints of late-stage human OA (Im et al. 2010). In line with these earlier reports, our histological and immunohistochemical findings indicate that synovitis is not a characteristic feature of the aging joint.

Animal models of OA have provided biological insights into OA-induced progressive pathological changes in knee joint structures (Im et al. 2010).

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Spontaneous OA is uncommon in rats (Gerwin et al.

2010), although minor foci of articular cartilage degeneration have been observed (Adams and Horton 1998; Gyarmati et al. 1987; Smale et al. 1995). Aging and the development of cartilage degeneration involve many factors, which either alone or in combination may accelerate the onset of OA (Grogan and D’Lima 2010). OA increases in prevalence with increasing age to becoming almost ubiquitous in elderly populations, making it difficult to be easily distinguished from agerelated changes (Mapp et al. 2008). However, our work emphasizes that it is important to understand the difference between the effects of natural aging and the manifestation of OA in joints. The changes seen in articular cartilage with aging followed a pattern similar to that of OA, while no sign of synovitis was observed. The extracellular matrix and cell functions of articular cartilage considered the most important factors in the development and progression of OA were found to change with aging. Although it is recommended that much older rats are used to mimic the development of OA in humans as closely as possible for all rat models of OA (Gerwin et al. 2010), future studies should require more attention to overestimating these severities. Also, prevalence of knee OA was higher in women than in men (Pereira et al.

2011). The female hormones have the potential to alter the properties of the periarticular connective tissues (Ohtera et al. 2002); therefore we used male rats.

Further study is needed to clarify whether similar results are expected in females or not.

The age-related changes seen in articular cartilage (with aging) were not abolished in our exercised old rats. This lack of cartilage recovery may be due to several factors, including the duration of the intervention, the regenerative capacity of the cartilage, and the small sample size. The exercise intervention period in the present study was identical to that of earlier studies investigating the effect of treadmill exercise training on physical performance and skeletal muscle apoptosis in old rats (Marzetti et al. 2008). Balance training can be effective for postural and neuromuscular control improvements (Zech et al. 2010). Muscle and neuromuscular improvements may take much longer to have a beneficial effect on age-related changes in the joint. With little or no potential for cartilage regeneration, additional research on this possibility is warranted. The statistical power of the present findings was partly reduced by the small sample size due to the

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half-mortality age of rats, which does not allow ruling out chance findings. Parameters with low statistical power are likely to generate/produce type II errors, necessitating larger sample sizes in future studies.

In conclusion, our study confirms that aging alone, as investigated in mechanical tests and by light microscopy, results in joint degeneration, eventually leading to OA, in a male rat model. Joint degeneration do not appear to be the inevitable consequence of aging, but instead is brought about by alterations in the aging joint that make it more susceptible to degeneration.

Acknowledgments This study was supported in part by Grant-in-Aid for Scientific Research (21500483) and for Young Scientists (21700545) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology. We thank Dr. Yoshiko Tobimatsu and Professor Seiichi Kawamata for their advice and expertise; Mr. Yoshio Shirasaki, Mr. Francis Terloo, Ms. Sofie Thys, and Mr. Dominique De Rijck for their skilled technical assistance; and Dr. Hidetaka Imagita and Mr. Tomoyuki Kurose for helpful discussions.

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