Moderate Physical Activity as a Prevention Method for Knee Osteoarthritis and the Role of Synoviocytes as Biological Key

Abstract

The purpose of this study was to investigate the influence of moderate physical activity (MPA) on the expression of osteoarthritis (OA)-related (IL-1β, IL-6, TNF-α, MMP-13) and anti-inflammatory and chondroprotective (IL-4, IL-10, lubricin) biomarkers in the synovium of an OA-induced rat model. A total of 32 rats were divided into four groups: Control rats (Group 1); rats performing MPA (Group 2); anterior cruciate ligament transection (ACLT)-rats with OA (Group 3); and, ACLT-rats performing MPA (Group 4). Analyses were performed using Hematoxylin & Eosin (H & E) staining, histomorphometry and immunohistochemistry. In Group 3, OA biomarkers were significantly increased, whereas, IL-4, IL-10, and lubricin were significantly lower than in the other experimental groups. We hypothesize that MPA might partake in rescuing type B synoviocyte dysfunction at the early stages of OA, delaying the progression of the disease.

Keywords: interleukins; lubricin; osteoarthritis; physical activity; synovium.

Figure 1

Histological evaluation and histomorphometric analysis of articular cartilage and synovium by Hematoxylin & Eosin (H&E) staining. (A) Group 1. Cells flat and small in the superficial zone; chondrocytes in columns in the middle and deep zone; tidemark evident; cartilage thickness 383.0 ± 76.76 µm. (B) Group 2. Numerous and small cells in the superficial zone; chondrocytes in columns in the middle and deep zone; tidemark evident; cartilage thickness 396.5 ± 61.68 µm. (C) Group 3. Articular cartilage showing structural alterations in the superficial and the middle zones and chondrocytes poorly organized in columns in the intermediate and deep zone; cartilage thickness 260.7 ± 62.34 µm. (D) Group 4. Articular cartilage showing only a slight reduction of the total thickness 357.9 ± 61.88 µm and a better general tissue preservation. (E) Graphs representing measures of the cartilage thickness, the Kraus’ Modified Mankin Score and the Histopathology OARSI System identified among groups. Results were presented as the mean ± SD. ANOVA was used to evaluate the significance of the results. * p < 0.05; ** p < 0.01; **** p < 0.0001; ns, not significant. For details, see the text. (F) H&E staining of synovium of Group 1. G H&E staining of synovium of Group 2. H H&E staining of synovium of Group 3. I H&E staining of synovium of Group 4. No histological alterations were evidenced in the synovium area of all groups. (A–D,F–I): Objective lens, 10×; scale bars: 100 µm.

Figure 2

(A–E): IL-1β immunohistochemistry. (A) IL-1β immunolabeling was very weak in the synovium of Group 1; (B) IL-1β immunolabeling was very weak in the synovium of Group 2; (C) IL-1β immunolabeling was moderate in the synovium of Group 3; (D) IL-1β immunolabeling was weak in the synovial membrane of Group 4; (E) Graph representing the densitometric count (Log2 densitometric count − pixel²) of IL-1-immunolabeling identified among groups. For details, see the text. (F–J): IL-4 immunohistochemistry. (F) IL-4 immunolabeling was moderate in the synovium of Group 1; (G) IL-4 immunolabeling was strong in the synovium of Group 2; (H) IL-4 immunolabeling was weak in the synovium of Group 3; (I) IL-4 immunolabeling was strong in the synovial membrane of Group 4; (J) Graph representing the densitometric count (Log2 densitometric count − pixel²) of IL-4-immunolabeling identified among groups. For details, see the text. (K–O): IL-6 immunohistochemistry. (K) IL-6 immunolabeling was moderate in the synovium of Group 1; (L) IL-6 immunolabeling was strong in the synovium of Group 2; (M) IL-6 immunolabeling was very strong in the synovium of Group 3; (N) IL-6 immunolabeling was very strong in the synovial membrane of Group 4; (O) Graph representing the densitometric count (Log2 densitometric count − pixel²) of IL-6-immunolabeling identified among groups. For details, see the text. (P–T): IL-10 immunohistochemistry. (P) IL-10 immunolabeling was moderate in the synovium of Group 1; (Q) IL-10 immunolabeling was strong in the synovium of Group 2; (R) IL-10 immunolabeling was weak in the synovium of Group 3; (S) IL-10 immunolabeling was strong in the synovial membrane of Group 4; and, (T) Graph representing the densitometric count (Log2 densitometric count − pixel²) of IL-10-immunolabeling identified among groups. For details, see the text. In inserts are the image analyses by the software in which red color represents immunolabeling. (A–D,F–I,K–N,P–S): Objective lens, 20×; scale bars: 50 µm. Results were presented as the mean ± SD. ANOVA was used to evaluate the significance of the results. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant.

Figure 3

(A–E): TNF-α immunohistochemistry. (A) TNF-α immunolabeling was weak in Group 1; (B) TNF-α immunolabeling was weak in Group 2; (C) TNF-α-immunostaining was strong in Group 3; (D) TNF-α-immunostaining was weak in Group 4; (E) Graph representing the densitometric count (Log2 densitometric count − pixel²) of TNF-α-immunolabeling identified among groups. For details, see the text. (F–J): MMP-13 immunohistochemistry. (F) In Group 1, MMP-13 was very weak; (G) In Group 2, MMP-13 was very weak; (H) MMP-13-immunostaining was strong in Group 3; (I) MMP-13-immunostaining was weak in Group 4; (J) Graph representing the densitometric count (Log2 densitometric count − pixel²) of MMP-13-immunolabeling identified among groups. For details, see the text. (K–O): Lubricin immunohistochemistry. (K) Lubricin immunolabeling was strong in the synovium of Group 1; (L) Lubricin immunolabeling was very strong in the synovium of Group 2; (M) Lubricin immunolabeling was moderate in Group 3; (N) Lubricin immunolabeling was strong in the synovial membrane of Group 4; and, (O) Graph representing the densitometric count (Log2 densitometric count − pixel²) of Lubricin-immunolabeling identified among groups. For details, see the text. In inserts are the image analyses by the software in which red color represents immunolabeling. (A–D,F–I,K–N): Objective lens, 20×; scale bars: 50 µm. Results were presented as the mean ± SD. ANOVA was used to evaluate the significance of the results. ** p < 0.01; **** p < 0.0001; ns, not significant.

Figure 4

Graphical representation of a joint affected by moderate osteoarthritis (OA) in sedentary people and in active people with relative benefits and disadvantages. Pro-inflammatory cytokines play a key role in the pathogenesis of OA, by mediating the progressive degeneration of the articular cartilage within the joint. The deteriorating processes involve different types of cells including macrophages, chondrocytes and synoviocytes.

Figure 5

Surgical procedures for anterior cruciate ligament transection (ACLT) step by step. (A) Pre-operative examinations and weighing of the rat; (B) Anesthesia of the rat; (C) Shearing of the rat; (D) Cutting the skin of the rat; (E) Cutting the capsule of the rat; (F) The patella was displaced laterally to expose the anterior cruciate ligament, then, the anterior cruciate ligament was cut with surgical scissors; (G) The skin around the knee cap was vertically incised along the medial border of the knee cap; (H) The patella was then replaced back, and the fascia and skin were closed with a 3–0 polydioxanone suture; (I) The rat after the suture under anesthesia before awakening; (J) The rat in the heated bed before awakening from anesthesia; (K) After surgery, free cage movement without joint immobilization was permitted to the rat; (L) Rats from Group 3 during exercise on Treadmill; (M) Knee joint covered to capsule; (N) Capsule removal; and, (O) Knee joint without capsule.