วารสารสัตวแพทยศาสตร์ มข. ปีที่ 22 ฉบับที่ 2 กรกฎาคม - ธันวาคม 2555 107 Catabolism of Asian Elephant Cartilage Matrix Biomolecules in Explant Culture Siriwan Tangyuenyong 1 , Nawarat Viriyakhasem 2 , Sirinda Aungsuchawan 3 , Siriporn Peansukmanee 1 , Chatchote Thitaram 1 , Prachya Kongtawelert 2 , Siriwan Ongchai 2* Abstract Objective—To attempt to culture and study articular cartilage biomolecules in Asian elephant. Materials and Methods—Elephant articular cartilages were dissected from knee joint. The explants were then incubated in culture medium containing antibiotics in a humidified incubator with 5% CO 2 at 37°C for 3-28 days, under condition of 10 ng/ml IL-1b-induced cartilage degradation, compare to left untreated as control. At the end of incubation, the conditioned media were collected to measure the quantity of hyaluronan (HA), sulfated glycosaminoglycan (s-GAG) and the matrix metalloproteinase-2 (MMP-2) activity by competitive inhibition-based-ELISA, colorimetric dye binding assay and gelatin zymography respectively. The remaining of collagen and uronic acid content in cartilage tissue were investigated by colorimetric dye binding assays. Results—IL-1b treated group showed 74% higher HA release than control (P<0.05) but there was no statistical difference in s-GAG release. MMP-2 activity was found to be 71% lower in IL-1b treated group than in control. The remaining of collagen and uronic acid in the explant tissues tended to be greater in the IL-1b treated group when compared to the control. The explant tissue section stained with Hematoxylin-Eosin and Safranin-O showed no significant difference in chondrocyte number and matrix profile; however, cell size in IL-1b treated group was likely to be bigger than that in the control. Conclusion—The achievement on cartilage explants culture and monitoring the marker substance of ar- ticular cartilage degradation provides the academic significance for further investigation of osteoarthritis in elephants. KKU Vet J. 2012;22(2):107-123. http://vmj.kku.ac.th/ Keywords: Elephant; Explant culture; Articular cartilage degradation; Osteoarthritis; IL-1b 1 Department of Companion Animal and Wildlife Clinic, Faculty of Veterinary Medicine, Chiang Mai University, Muang, Chiang Mai, 50100 2 Thailand Excellent Center for Tissue Engineering and Stem Cells, Department of Biochemistry and Center of Excellence for Innovation in Chemistry, Faculty of Medicine, Chiang Mai University, Muang, Chiang Mai, 50200 3 Department of Anatomy, Faculty of Medicine, Chiang Mai University, Muang, Chiang Mai, 50200 * Corresponding author E-mail: [email protected]RESEARCH ARTICLE
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Objective—To attempt to culture and study articular cartilage biomolecules in Asian elephant.
Materials and Methods—Elephant articular cartilages were dissected from knee joint. The explants were then incubated in culture medium containing antibiotics in a humidified incubator with 5% CO
2
at 37°C for 3-28 days, under condition of 10 ng/ml IL-1b-induced cartilage degradation, compare to left untreated as control. At the end of incubation, the conditioned media were collected to measure the quantity of hyaluronan (HA), sulfated glycosaminoglycan (s-GAG) and the matrix metalloproteinase-2 (MMP-2) activity by competitive inhibition-based-ELISA, colorimetric dye binding assay and gelatin zymography respectively. The remaining of collagen and uronic acid content in cartilage tissue were investigated by colorimetric dye binding assays.
Results—IL-1b treated group showed 74% higher HA release than control (P<0.05) but there was no statistical difference in s-GAG release. MMP-2 activity was found to be 71% lower in IL-1b treated group than in control. The remaining of collagen and uronic acid in the explant tissues tended to be greater in the IL-1b treated group when compared to the control. The explant tissue section stained with Hematoxylin-Eosin and Safranin-O showed no significant difference in chondrocyte number and matrix profile; however, cell size in IL-1b treated group was likely to be bigger than that in the control.
Conclusion—The achievement on cartilage explants culture and monitoring the marker substance of ar-ticular cartilage degradation provides the academic significance for further investigation of osteoarthritis in elephants.
KKU Vet J. 2012;22(2):107-123. http://vmj.kku.ac.th/
Keywords: Elephant; Explant culture; Articular cartilage degradation; Osteoarthritis; IL-1b1Department of Companion Animal and Wildlife Clinic, Faculty of Veterinary Medicine, Chiang Mai University, Muang, Chiang Mai, 50100 2Thailand Excellent Center for Tissue Engineering and Stem Cells, Department of Biochemistry and Center of Excellence for Innovation in Chemistry, Faculty of Medicine, Chiang Mai University, Muang, Chiang Mai, 50200 3Department of Anatomy, Faculty of Medicine, Chiang Mai University, Muang, Chiang Mai, 50200 *Corresponding author E-mail: [email protected]
เปรยบเทยบลกษณะทพบในกลม normal culture กบกลม IL-1b induced culture ปรากฏวาไมมความ
แตกตางกน ดงแสดงใน Figure7d,7eและ7f ตามล�าดบ
Figure 1. IL-1b induced degradation of cartilage matrix biomolecules of the Asian elephant
articula cartilage explants
Elephant articular cartilage explants were treated with IL-1b at concentration 10 ng/ml or left untreated as control. After 3 days of incubation, the explants culture were harvested for analysis the release of cartilage matrix biomolecules into the culture media such as HA and s-GAG. The cartilage tissues were analyzed for uronic acid content. The results are expressed as a percentage relative to the control. *Denoted value that was significant different (P<0.05) from the control.
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Figure 2. Zymographic analysis of MMP-2 activity from the culture medium of Asian elephant
articular cartilage explants induced by IL-1b (a) and optical density values of MMP-2 activity
relative to the control group (b).
66 kDa a
b
Elephant articular cartilage explants were treated with IL-1b at concentration 10 ng/ml or left untreated as control. After 3 days of incubation, the culture media were analyzed for the gelatinolytic activity of MMP-2. Density of the clear bands which represented the gelatinase activity is expressed in the bar graph as relative to the control.
Figure 3 . Cytotoxicity effect of IL-1b on Asian elephant articular cartilage explants measuring
by LDH assay
Elephant articular cartilage explants were treated with IL-1b at concentration 10 ng/ml or left untreated as control. After 3 days of incubation, the culture media were analyzed for the release of LDH. The positive control treated with 10 mM of H2O2 showed significant difference from the control (** = P <0.01).
Figure 5. Effect of IL-1b on the release accumulation of s-GAG to culture medium from Asian
elephant articular cartilage explants
Elephant articular cartilage explants were cultured for 28 days under treatment with IL-1b at concentration 10 ng/ml or left untreated as control. The culture media were changed at day 4, 7, 14, 21 and 28 to analyze for s-GAG accumulative concentration.
Figure 4 . Effect of IL-1b on the release accumulation of HA to culture medium from Asian
elephant articular cartilage explants
Elephant articular cartilage explants were cultured for 28 days under treatment with IL-1b at concentration 10 ng/ml or left untreated as control. The culture media were changed at day 4, 7, 14, 21 and 28 to analyze for HA accumulative concentration.
KKU Vet J Vol. 22 No. 2 July - December 2012116
Figure 7. Histological staining of articular cartilage of the Asian elephant (Elephas maximus)
Fresh elephant articular cartilage tissues, the 28 days explants treated with IL-1b treated (+/IL-1b) and the untreated control (-/IL-1b) were proceeded sections and stained with Hematoxylin Eosin (a-c) and Safranin-O, the staining used for the contents of acidic glycosaminoglycans (d-f). All sections were examined under a light microscope. Bar = 100 µm.
Figure 6. Effect of IL-1b on the remaining of uronic acid and collagen contents in Asian
elephant articular cartilage explants
Elephant articular cartilage explants were treated with IL-1b at concentration 10 ng/ml or left untreated as control. The cartilage tissues were harvested at day 28 to analyze for uronic acid and collagen contents. The results are expressed as a percentage relative to the control. There was no significant different between the IL-1b treated and control (P<0.05).
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