Effects of ascorbic acid on proliferation and biological properties of bovine chondrocytes in alginate beads.
Bovine chondrocytes were cultured in monolayers and alginate beads with or without ascorbic acid (Asc) for 16 days. Cell proliferation was examined every 4 days by staining with Hoechst 33258 dye. The gene expression of aggrecan, and collagen type I and II was analyzed at 16 days by reverse transcription and polymerase chain reaction. Cell morphology and the production of extracellular matrix (ECM) were evaluated by cytochemical, immunocytochemical and electron microscopical methods. Cells were continuously cultured in alginate beads with Asc for 2 months, and the cell morphology and ECM were examined. The proliferation of chondrocytes was significantly stimulated with Asc in both monolayers and alginate beads at 16 days. Expression of the collagen type I gene in both cultures was increased, and that of the collagen type II gene in alginate beads was decreased, by Asc. There were no significant cytochemical and immunocytochemical differences between the cultures in alginate beads with or without Asc at 16 days. In alginate beads cultured with Asc for 2 months, proliferating cells were observed mainly at the periphery of the beads, and glycosaminoglycan and collagen type II were found around the cells. These results suggest that Asc stimulated the proliferation of chondrocytes and maintained the chondrogenic properties of the cells in an alginate beads culture.
Kim
Gonhyung
G
Laboratories of Veterinary Surgery, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan. vetkim@vetmed.hokudai.ac.jp
Okumura
Masahiro
M
Bosnakovski
Darko
D
Ishiguro
Taketo
T
Park
Chun-Ho
CH
Kadosawa
Tsuyoshi
T
Fujinaga
Toru
T
D053674:Aggrecans; D000464:Alginates; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D001205:Ascorbic Acid / Q000494:pharmacology*; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism*; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects* / Q000502:physiology / Q000648:ultrastructure; D003094:Collagen / Q000096:biosynthesis / Q000235:genetics; D004247:DNA / Q000032:analysis; D005109:Extracellular Matrix / Q000378:metabolism; D016326:Extracellular Matrix Proteins; D015870:Gene Expression; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D006651:Histocytochemistry; D007150:Immunohistochemistry / Q000662:veterinary; D037181:Lectins, C-Type; D008854:Microscopy, Electron; D008863:Microspheres; D011509:Proteoglycans / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary
D053674:Aggrecans; D000464:Alginates; D000975:Antioxidants; D016326:Extracellular Matrix Proteins; D006603:Hexuronic Acids; D037181:Lectins, C-Type; D011509:Proteoglycans; D020723:Glucuronic Acid; D003094:Collagen; D004247:DNA; D001205:Ascorbic Acid