Anat Cell Biol.  2019 Dec;52(4):498-510. 10.5115/acb.19.050.

Protective effect of glucosamine and risedronate (alone or in combination) against osteoarthritic changes in rat experimental model of immobilized knee

• Salman A
• Shabana AI
• El-ghazouly DEs
• Maha E

• KMID: 2466704
• DOI: http://doi.org/10.5115/acb.19.050

Abstract

This study is aiming to investigate the protective effect of glucosamine, risedronate (alone or in combination) on articular cartilage in experimental model of immobilized rat knee. Twenty-five adult male albino rats were divided into five groups (five rats each): control group, immobilized group, glucosamine-treated group, risedronate-treated group, and group treated by a combination of glucosamine and risedronate. The articular cartilage was obtained for histological, immunohistochemical and morphometric studies. The immobilized group showed manifestations of osteoarthritis in the form of significant decrease of articular cartilage thickness with surface erosions, shrunken chondrocytes with pyknotic nuclei and marked manifested fall of chondrocyte number. There was manifested reduction of collagen contents of the articular cartilage using Masson trichrome stain. Safranin O-Fast Green revealed low proteoglycan contents. The collagen type II was also declined. The manikin score was 7.8. Risedronate improved this manifestation slightly more than glucosamine, but combination of booth drugs caused significant improvement of the damaged articular cartilage caused by immobilization. Oral administration of glucosamine and risedronate improved the degenerative changes of rat knee articular cartilage that follow immobilization. This improvement was more remarkable when both drugs were used in combination.

Keyword

Knee; Immobilization; Glucosamine; Risedronate; Osteoarthritis

MeSH Terms

Administration, Oral
Adult
Animals
Cartilage, Articular
Chondrocytes
Collagen
Collagen Type II
Glucosamine*
Humans
Immobilization
Knee*
Male
Manikins
Models, Theoretical*
Osteoarthritis
Proteoglycans
Rats*
Risedronate Sodium*
Collagen
Collagen Type II
Glucosamine
Proteoglycans
Risedronate Sodium

Figure

• 

  Fig. 1 Hematoxylin and eosin (H&E)–stained sections of the knee joint of different groups. (A) Control group showing articular cartilage with regular smooth intact surface and well-organized chondrocytes which appeared in non-calcified (NCC) and calcified (CC) regions of cartilage with a clear intact tidemark (arrows) in between. The subchondral bone (SC) appears intact. The meniscus shows regular smooth surface with no fraying or undulation. (B) Immobilized group showing reduction in thickness of articular cartilage, shrunken chondrocytes, absence of chondrocytes in some areas (*) and invisible tidemark. The SC appears intact. Meniscus shows severe fraying and tears and erosion of surface with necrotic cells shedding from it (arrow). (C) The immobilized group treated with glucosamine; the articular cartilage shows smooth surface, shrunken chondrocytes and visible tidemark (*). The SC appears intact. The meniscus shows smooth surface with some cracks (arrow). (D) The immobilized group treated with risedronate; the articular cartilage shows smooth surface, shrunken chondrocytes, some empty lacunae, and hardly visible tidemark. The SC appears intact. The meniscus shows smooth surface with some cracks (arrow). (E) immobilized group treated with glucosamine and risedronate; the articular cartilage shows smooth surface, few shrunken chondrocytes, some empty lacunae, and visible tidemark. The SC appears intact. The meniscus shows minimal erosion of its surface (arrow) (H&E, ×200). ar, articular cartilage; L, lacunae; m, meniscus; t, tears.

• 

  Fig. 2 Hematoxylin and eosin (H&E)–stained sections of articular cartilage of knee joint of different groups. (A) Control group showing the chondrocytes in non-calcified region (NCC) of the articular cartilage arranged in three zones: superficial (S), transitional (T), and radial (R) zone. The superficial zone contains small flat chondrocytes. The transitional and radial zone contain columns of rounded, oval, or triangular chondrocytes (*). The chondrocytes are located inside their lacunae forming cell nests. The calcified region (CC) is separated from radial zone by a tidemark (arrows). The subchondral bone (SC) appears intact. (B) Immobilized group; articular cartilage shows irregular notched surface (arrows) and its chondrocytes appear shrunken with pyknotic nuclei, disorganized and few in number. Tidemark is invisible. The subchondral bone shows degenerative changes (*). (C) The immobilized group treated with glucosamine; articular cartilage shows irregular degenerated surface (arrow) and shrunken chondrocytes which appear disorganized and few in number. Tidemark is visible. The SC appears intact. (D) The immobilized group treated with risedronate; articular cartilage shows smooth surface, shrunken chondrocytes which appear disorganized and few in number. Tidemark is not clearly visible. (E) The immobilized group treated with glucosamine and risedronate; articular cartilage shows smooth surface, few shrunken chondrocytes, few empty lacunae, and visible tidemark (arrow) (H&E, ×400). ch, chondrocyte; f, flat chondrocyte; L, lacunae; ne, cell nests.

• 

  Fig. 3 Hematoxylin and eosin–stained sections of meniscus of the knee joint of different groups. (A) Control group showing the meniscus composed of homogenous eosinophilic staining well-organized collagen fibers (arrows) with fibrochondrocytes in their lacunae. The meniscus surface is smooth with no fraying or undulation (*). (B) Immobilized group showing many meniscal tears with unorganized disrupted collagen fibers (arrows) and markedly shrunken darkly stained fibrochondrocytes. (C) The immobilized group treated with glucosamine showing some tears and cracks (arrows) and moderately shrunken darkly stained fibrochondrocytes. (D) The immobilized group treated with risedronate showing few tears and cracks (arrows) and slightly shrunken fibrochondrocytes. (E) The immobilized group treated with glucosamine and risedronate showing nearly normal meniscus except for minimal erosion of its surface (arrow) (H&E, ×400). fc, fibrochondrocytes.

• 

  Fig. 4 A photomicrograph of Masson trichrome–stained sections of articular cartilage of knee joint of different groups. (A) Control group showing the articular cartilage which is well stained with Masson trichrome for collagen (green color) (*). (B) Immobilized group showing marked reduction of Masson trichrome–stained area for collagen with appearance of an extensive red color (*). Minimal erosion of the articular cartilage surface is observed (arrow). (C) The immobilized group treated with glucosamine showing marked reduction of Masson trichrome–stained area for collagen with appearance of a red color (*). (D) The immobilized group treated with risedronate showing moderate reduction of Masson trichrome–stained area for collagen with appearance of a red color (*). (E) The immobilized group treated with glucosamine and risedronate showing slight reduction of Masson trichrome–stained area for collagen with appearance of a slight red color (*) (Masson trichrome, ×400).

• 

  Fig. 5 A photomicrograph of Safranin O–Fast Green (SO)–stained sections of articular cartilage of knee joint of different groups. (A) Control group showing the articular cartilage which appears well stained with SO. The smooth surface of the articular cartilage (arrow) with normal cellular distribution is observed. (B) Immobilized group showing marked reduction in SO staining intensity in the non-calcified region of the articular cartilage (*) and slight reduction in its calcified region. The surface of articular cartilage shows fibrillation (arrow). (C) The immobilized group treated with glucosamine showing marked reduction in SO staining intensity in the superficial part (arrow) of the articular cartilage and moderate reduction in its deeper part (*). The smooth surface of the articular cartilage is observed. (D) The immobilized group treated with risedronate showing moderate reduction in SO staining intensity in the calcified region of the articular cartilage (*) with disruption in the superficial part of the articular cartilage (arrow). (E) The immobilized group treated with glucosamine and risedronate showing slight reduction in SO staining intensity in the superficial part of the articular cartilage (*). The smooth surface of the articular cartilage is observed (SO staining, ×400).

• 

  Fig. 6 Immunohistochemical stained sections for collagen type II of the articular cartilage of the knee joint of different groups. (A) Control group showing very strong immunostaining intensity for collagen type II in articular cartilage (brown color). The smooth surface of the articular cartilage is observed. (B) Immobilized group showing weak immunostaining intensity for collagen type II in articular cartilage with disruption in the superficial part of cartilage (arrow). (C) The immobilized group treated with glucosamine showing moderate immunostaining intensity for collagen type II in articular cartilage (*). Irregularity of the surface (arrow) and presence of a space in superficial part are observed. (D) The immobilized group treated with risedronate showing moderate immunostaining intensity for collagen type II in articular cartilage (*) with necrosis of superficial part of the articular cartilage (arrow). (E) The immobilized group treated with glucosamine and risedronate showing strong immunostaining intensity for collagen type II in the articular cartilage. The smooth surface of the articular cartilage is observed (immunohistochemical staining for collagen type II, ×400). s, space.

• 

  Fig. 7 (A) The thickness of the articular cartilage (µm) of different experimental groups. Pairwise significant differences were detected between: group I and group II (P<0.001); group II and group III (P<0.05), group IV (P<0.01) and group V (P<0.001); group III and groups IV and V (P>0.05); group IV and group V (P>0.05). (B) The number of the chondrocytes in the articular cartilage of different experimental groups. Pairwise significant differences were detected between: group I and group II (P<0.001); group II and groups III, IV and V (P<0.001); group III and groups IV and V (P>0.05); group IV and group V (P>0.05). (C) The number of the chondrocytes in the articular cartilage of different experimental groups. Pairwise significant differences were detected between: group I and group II (P<0.001); group II and groups III, IV, and V (P<0.001); group III and group IV (P>0.05), V (P<0.001); group IV and group V (P<0.001). Group I, control; group II, immobilized knee; group III, immobilized group treated with glucosamine; group IV, immobilized group treated with risedronate; group V, immobilized group treated with glucosamine and risedronate. Analysis was done using GraphPad Prism version 4. Data were analyzed by using one-way analysis of variance test followed by Bonferroni's multiple comparison post-hoc test for comparison between all groups. P>0.05, nonsignificant; P<0.05, significant.

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