Sains Malaysiana 48(8)(2019): 1671–1683

http://dx.doi.org/10.17576/jsm-2019-4808-13

The Effect of Stichopus chloronotus Aqueous Extract on Human Osteoarthritis Articular Chondrocytes in Three-Dimensional Collagen Type I Hydrogel in vitro

(Kesan Ekstrak Akues Stichopus chloronotus pada Kondrosit Osteoartritis Artikul Manusia

dalam Kolagen Tiga Dimensi Hidrogel Jenis I secara in vitro)

MOHD HEIKAL MOHD YUNUS^1,4*, AHMAD NAZRUN SHUID², MOHD FAUZI BUSRA⁴, CHUA KIEN HUI¹, NORZANA ABDUL GHAFAR³ & RIZAL ABD RANI⁵

¹Department of Physiology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

²Department of Pharmacology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

³Department of Anatomy, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latiff, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

⁴Tissue Engineering Centre, Universiti Kebangsaan Malaysia Medical Center, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras Kuala Lumpur, Federal Territory, Malaysia

⁵Department of Orthopaedic, Universiti Kebangsaan Malaysia Medical Center, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras Kuala Lumpur, Kuala Lumpur, Federal Territory, Malaysia

Received: 25 September 2018/Accepted: 6 May 2019

ABSTRACT

Autologous chondrocyte-seeded scaffolds have proved to be one of the most promising alternative therapies for articular cartilage defects. However, the chondrocytes have specific nutritional requirements and risk of dedifferentiation during in vitro expansion. Stichopus chloronotus aqueous extract (SCAE) was investigated as a medium supplement for three-dimensional (3D) collagen type I hydrogel scaffold seeded with chondrocytes to determine whether SCAE is capable of maintaining phenotype and sustaining extracellular matrix synthesis and deposition. Human osteoarthritis articular chondrocytes were isolated from the knee joint cartilage of patients underwent total knee replacement surgery. Human osteoarthritis articular chondrocytes were encapsulated in collagen type I hydrogel and cultured in basic medium with 0, 0.1 and 0.2% of SCAE. The chondrocytes in 3D culture were evaluated by means cell morphology and proliferation, quantitative phenotypic expression of collagen type I, II, aggrecan core protein and sox-9. H&E, toluidine blue staining and sulfated glycosaminoglycan (sGAG) production were analyzed after 7 days in culture. Chondrocytes cultured in 3D with various SCAE concentration appeared with polygonal morphology maintaining their chondrocytes characteristic. SCAE supplementation promoted chondrocytes proliferation and the ability of the cells to express gene encoding collagen type I decreased, whereas their ability to express collagen type II, aggrecan core protein and sox9 increased as compared to control. The cartilaginous matrices were positively stained toluidine blue concomitant with higher sGAG accumulation in SCAE-supplemented culture medium. This study shown that SCAE may be beneficial for in vitro development of 3D chondrocytes culture for use in cartilage tissue engineering therapies.  

Keywords: Chondrocytes; collagen Type I; osteoarthritis; Stichopus chloronotus; three dimensional culture

ABSTRAK

Biobahan yang disemai kondrosit secara autologus telah terbukti menjadi salah satu terapi alternatif yang paling berkesan untuk kecacatan rawan artikul. Walau bagaimanapun, kondrosit mempunyai keperluan nutrien tertentu dan berisiko untuk mengalami nyahpembezaan semasa pengembangan in vitro. Ekstrak air Stichopus chloronotus (SCAE) telah dikaji sebagai tambahan bagi media untuk kondrosit yang disemai dalam hidrogel kolagen jenis I secara tiga dimensi (3-D) untuk menentukan sama ada SCAE mampu mengekalkan fenotip dan sintesis serta pemendapan matriks ekstrasel. Kondrosit osteoartritis manusia dipencilkan daripada rawan sendi lutut pesakit yang menjalani pembedahan penggantian lutut secara total. Kondrosit osteoartritis manusia disemai dalam hidrogel kolagen jenis I dan dikultur dalam medium asas dengan 0, 0.1 dan 0.2% SCAE. Kondrosit yang dikultur secara 3D dinilai daripada segi morfologi dan percambahan sel, ekspresi fenotip kuantitatif bagi kolagen jenis I, II, protein teras agrekan dan sox-9. Pewarnaan H&E dan toluidin biru serta penghasilan glikosaminoglikan tersulfat (sGAG) dianalisis selepas dikultur selama 7 hari. Kondrosit yang dikultur secara 3D dengan pelbagai kepekatan SCAE menunjukkan morfologi poligonal mengekalkan ciri-ciri kondrosit. Penambahan SCAE meningkatkan percambahan kondrosit dan keupayaan sel untuk mengekspresi gen pengekod kolagen jenis I menurun, sedangkan keupayaan untuk mengekspresikan gen kolagen jenis II, protein teras agrekan dan sox9 meningkat berbanding dengan kawalan. Matriks ekstrasel diwarnakan positif oleh toluidin biru, setara dengan pengumpulan sGAG yang lebih tinggi dalam media kultur dengan penambahan SCAE. Kajian ini memperlihatkan bahawa SCAE boleh memberi manfaat dalam pembentukan kultur kondrosit 3D secara in vitro untuk digunakan dalam terapi kejuruteraan tisu rawan.

Kata kunci: Kolagen jenis I; kondrosit; kultur tiga dimensi; osteoarthritis; Stichopus chloronotus

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*Corresponding author; email: mohdheikalyunus@yahoo.com