Age-related changes in rat joint capsule

Abstract

[Purpose] We investigated morphological and histopathological changes in the joint capsule of rats with aging. [Materials and Methods] A total of 18 male Wistar rats were categorized into two groups: the control group (n=8), and the aged group (n=10). The aged group was reared until 75 weeks of age, while the control group was maintained until 11 weeks of age. At the end of the experiment period, the knee joints were sampled, joint capsules were subjected to histopathological analysis, and their thickness was measured. [Results] The joint capsule in the aging group exhibited significantly greater thickness compared to the control group. Histopathological examination revealed distinct differences between the two groups. The control group displayed gaps between the collagen fibers in the posterior joint capsule, along with loosely overlapping connective tissue and the presence of fat cells. Conversely, in the aged group’s joint capsule, these gaps between the collagen fibers almost disappeared and fibers became densely packed and thickened. [Conclusion] These results were similar to our previous study in rats with immobilized hindlimb knee joints. Similar findings, including collagen fiber thickening, densification in the joint capsule, and reduced hindlimb knee joint range of motion, were consistent with those observed in the present investigation.

INTRODUCTION

It is very important for people to have a sufficient range of motion in their joints to lead their daily lives. However, it has long been known that joint range of motion decreases with age, and there have been many reports investigating the range of motion of young and elderly people.

Hageman and Blanke¹⁾ found that the ankle joint range of motion is smaller and walking speed is slower in the elderly, Einkauf et al.²⁾ found that the spine range of motion is decreased in the elderly, and Roach and Miles³⁾ found that the hip and knee joints range of motion is decreased in the elderly, all suggesting that aging has some effects on the joint components.

Osteoarthritis, a disease that changes joint components, occurs with aging and numerous studies have been reported on this. Osteoarthritis onset is directly proportional to age⁴⁾ where degenerative changes in the articular cartilage, synovial membrane inflammation, meniscus degeneration, and joint capsule enlargement are believed to occur⁵⁾.

In a study of age-related changes in joint range of motion other than osteoarthritis, Matsumura et al.⁶⁾ found that range of motion was significantly greater in the young group than in the middle-aged and elderly groups, except in hip and knee joint flexion and ankle joint dorsiflexion. Bok et al.⁷⁾ states that a decrease in ankle joint range of motion and muscle strength causes a decrease in balance in the elderly. However, most of these studies examined age-related range of motion limitation by measuring joint range of motion during physical activity, but none of them conducted a histological examination of the reasons for the limitation of range of motion.

It has been known that joint capsule release, either surgically or arthroscopically, can improve the joint range of motion limitation following elbow trauma^8,9,10). This suggested that the joint capsule may influence joint range of motion limitation, but there have been no reports of histological studies on age-related changes in the joint capsule.

In this study, a rat aging model was created to observe the effects of aging on the joint capsule.

MATERIALS AND METHODS

The Animal Research Committee of the Graduate School of Medicine of Kanazawa University (Kanazawa, Japan; Approval No. 183986) approved the study protocol and conducted in accordance with the ARRIVE guidelines¹¹⁾, guidelines for the animal care and relevant laws and regulations.

This study used 9-week-old male Wistar rats (n=18). Rats were purchased from a supplier (Sankyo Labs, Toyama, Japan) and kept in an animal house at the author’s institution. All rats were individually caged after purchase, with the aged group (n=10) reared to 75 weeks of age and the control group (n=8) to 11 weeks of age. Both groups were reared without intervention during the rearing period. During the rearing period, lighting was switched on and off every 12 h and the rearing room was maintained at 21°C–24°C. Fresh food and water were provided without restriction and care was taken to maintain a clean environment during rearing.

At the end of the rearing period, the rats were weighed, euthanized, and their right hind limbs were disarticulated from the hip joint. The collected lower extremities were subjected to tissue fixation using 4% neutral buffered formalin (FUJIFILM Wako Pure Chemical Co., Tokyo, Japan), followed by decalcification using the Plank–Rychlo solution. The knee joint was divided into two sections in the sagittal plane and neutralized with a 5% sodium sulfate solution after decalcification was completed.

Tissue fixation, decalcification, and neutralization were each set for 72 h, although 96 h was used because of insufficient decalcification in the aged group. All treatments were performed at 4°C. Specimens were embedded in paraffin and cut into 3-μm thick sections. The observation site was the plane through the middle of the medial femoral condyle. Specimens were stained with haematoxylin-eosin staining and immunostaining with anti-type 1 collagen antibody.

Tissue slides were deparaffinized, hydrated with graded alcohol, and washed with phosphate buffered saline (PBS) for 5 min. The sections were placed in PBS and subjected to antigen retrieval for 60 min in a thermostatic chamber maintained at 75°C, and then left at room temperature to cool slowly. Endogenous peroxidase block was then performed in PBS containing 3% hydrogen peroxide for 20 min at room temperature. Protein blocking was performed at room temperature for 10 min using Blocking One Histo (Nacalai Tesque, Inc., Kyoto, Japan). Between each step, the slide were rinsed twice with PBS for 3 min. The sections were incubated with antibodies Anti-Collagen I antibody (1:400, ab245113; Abcam plc, Cambridge, UK) overnight at 4°C.

The sections were then incubated with secondary antibody (Histofine^® Simple StainTM Rat MAX PO (R), Nichirei Biosciences Inc., Tokyo, Japan) for 60 min at room temperature. After rinsing, Histofine DAB Substrate Kit (Nichirei Biosciences Inc.) was prepared according to instructions and dropped onto the sections for staining. After staining, haematoxylin staining was performed as a counterstain and sealed. All sections were stained at the same time and the coloration time was identical.

An optical microscope (BX-51, Olympus Co., Tokyo, Japan) was used to observe the immunostained sections and a digital camera (DP74, Olympus Co., Tokyo, Japan) was used to photograph the posterior knee joint capsule. The midpoint of the line segment connecting the two ends of the arthrodesis just below the meniscus was determined and the joint capsule thickness was measured on a perpendicular line from that site to the outside of the joint capsule (Fig. 1.) with ImageJ (Ver.1.53n).

Fig. 1.

Sagittal section of knee joint capsule of rats. The line segment connecting the rat’s joint capsule was determined just below the meniscus, as shown in the figure. The vertical line from the midpoint to the posterior joint was measured as the joint capsule’s thickness (white arrow). F: femur; T: tibia; M: meniscus. Immunostaining with anti-type 1 collagen antibody. Scale bar: 500 μm.

The articular cartilage and meniscus were removed from the photographed posterior joint capsule (Fig. 2A) using Adobe Photoshop CS5.1 for Windows (Adobe Inc., San Jose, CA, USA) and converted to 8-bit grayscale (Fig. 2B). This was done while checking images by haematoxylin-eosin staining and immunostaining. ImageJ was used to measure the stained area (Fig. 2C) and the entire joint capsule area (Fig. 2D). In addition, the thresholds for the measurements were preset values, which were used to calculate the stained area and the entire joint capsule area, respectively.

Fig. 2.

Measurement of tissue gaps in the joint capsule. HE-stained image of joint capsule (A) with the meniscus, and articular cartilage removed and grayscaled (B). Stained area (C) and entire joint capsule (D) incorporated into ImageJ.

The percentage of tissue gaps of joint capsule was calculated as (1− staining area / entire joint capsule area) × 100 (%).

The stained area was divided by the area of the entire joint capsule to calculate the percentage of tissue gaps in the joint capsule.

Joint capsule thickness and tissue gap measurement were measured by one blinded examiner.

Statistical analyses (t-test with two independent variables) were performed by using SPSS Statistics (Ver. 25.0.0.2, IBM Corp, Armonk, NY, USA) with a significance level of <5%.

RESULTS

The control group’s mean body weight was 346.1 ± 15.0 g (mean ± standard deviation), and the aged group was 576.0 ± 43.5 g, showing a significant difference between the two groups (p<0.01). The joint capsule thickness in the control group was 535.7 ± 162.8 μm and the aging group was 1,003.4 ± 460.2 μm, with a significantly increased joint capsule thickness in the aging group (p=0.017) (Table 1).

Table 1. Comparison of aged group and control group.

	Control group (n=8)	Aged group (n=10)	p
Body weight (g)	346.1 ± 15.0	576.0 ± 43.5	<0.01
Joint capsule thickness (μm)	535.7 ± 162.8	1,003.4 ± 460.2	0.017
Percentage of tissue gaps in the joint capsule (%)	29.7 ± 10.9	15.5 ± 7.3	0.004

The mean area of staining sites was 358,426.7 ± 78,905.8 μm² in the control group and 777,087.1 ± 202,259.2 μm² in the aging group, and the mean entire joint capsule area was 511,801.8 ± 90,036.4 μm² in the control group and 973,074.1 ± 142,024.9 μm² in the aging group. The percentage of tissue gaps in the joint capsule, in the control group was 29.7 ± 10.9% and the aging group was 15.5 ± 7.3%, with a significantly decreased percentage of tissue gaps in the aging group (p=0.004).

Histopathological findings showed that the posterior joint capsule in the control group (Fig. 3A) had gaps between collagen fibers and the loosely overlapping connective tissue and fat cells were observed in the joint capsule (Fig. 3B). In the aged group’s joint capsule (Fig. 3C), the gaps between the collagen fibers almost disappeared and fibers became densely grown and thickened, and the number of fat cells decreased or were not observed (Fig. 3D).

Fig. 3.

Histopathological changes in the joint capsule. The control group had gaps between the collagen fibers and loosely overlapping connective tissue (A), and fat cells were observed in the joint capsule (B). Meanwhile, in the aged group (C), collagen fibers became densely proliferated and thickened, and the number of fat cells was reduced or no longer observed (D). Scale bar=Fig. 3A, 3C: 500 μm; Fig. 3B, 3D: 200 μm.

DISCUSSION

The joint capsule is composed of dense connective tissue, mainly composed of type I collagen, and is tightly attached to the periosteum. Synovium is present on the joint cavity’s medial surface outside the articular cartilage. The synovial tissue consists of a synovial intima composed of 0–3 layers of synovial surface cells and contains extracellular matrix such as fibronectin, tenascin, laminin, entactin, and type III collagen, but no basal membrane. Immediately beneath is a subsynovial tissue composed of vascular-rich connective tissue or fatty tissue. The synovial tissue is continuous into the joint capsule without defined boundaries. Although adipocytes were observed in the joint capsule in this study, to the best of our knowledge, no histological examination of the joint capsule, including the presence or absence of adipocytes, has been performed.

In our previous study^{12, 13)} in rats with immobilized hindlimb knee joints, the same findings were observed as in the present study, such as collagen fiber thickening and densification in the joint capsule, as well as decreased hindlimb knee joint range of motion, indicating that joint motion loss causes fibrosis and thickening of the joint capsule and may be a cause of joint contracture.

In this study, collagen fiber densification, inter-fiber gap narrowing, and entire joint capsule thickening due to fibrosis in the joint capsule of aged animals were observed. These results were similar to those of the posttraumatic joint contractures and the joint immobilization models.

The joint capsule can be damaged and develop adhesions to surrounding structures¹⁴⁾. Hildebrand et al.^15,16,17) developed a posttraumatic joint contracture model, which described the relationship between joint capsule and joint contractures. They stated that joint contractures develop due to thickening and joint capsule fibrosis, the joint capsule tissue undergoes metabolic renewal, and joint capsule fibrosis is caused by myofibroblasts that are produced during the healing process of trauma.

In recent years, the concept of understanding age-related and lifestyle-related diseases, such as chronic inflammation had been proposed based on the observation of increased inflammatory markers and activation of inflammatory signals in the elderly^18,19,20). Diseases that cause fibrosis of living tissue include pulmonary fibrosis and liver cirrhosis. Aging is thought to be a factor in pulmonary fibrosis pathogenesis and these diseases are characterized by fibroblast proliferation presence in the tissues as a pathological finding. Fibroblasts are connective tissue cells that are ubiquitously distributed in vivo and produce procollagen as a secreted product. Procollagen becomes collagen molecules outside the cell and converges to form collagen fibrils. Fibroblasts are also involved in wound healing, during which they differentiate into myofibroblasts, which contract the wound and form a scar. The present results suggest that fibroblast hyperplasia may have occurred as a healing process of chronic inflammation caused by aging, resulting in collagen fiber bundle densification, fibrosis, and joint capsule thickening.

This study’s limitation was that the aging group was kept in cages for a longer period, which may have reduced their locomotion. Regarding the relationship between exercise and joint range of motion in the elderly, Misner et al.²¹⁾ found that weekly exercise improved or maintained range of motion in the elderly, and Zakas et al.²²⁾ found that teaching the elderly to stretch for short periods of time resulted in increased joint range of motion.

We did not evaluation the physical activity and joint movements in this study, and histological changes in the joint capsule and other components due to exercise loading should be examined in the future, although it has been suggested that aging and/or exercise has some effect on joint components as described above.

The aged animals’ joint capsule was significantly thickened compared to the young controls, with dense collagen fibers and inter-fiber space loss. These histological images were like those of the joint capsule observed in the joint contracture model.

Funding

This research was supported by JSPS KAKENHI Grant Number 19K11413.

Conflict of interest

There are no conflicts of interest to declare. The authors are solely responsible for the content of the paper.