Effect of different oxygen tension on the cytoskeleton remodeling of goat temporomandibular joint disc cells

doi:10.7518/hxkq.2017.04.004

Abstract

Abstract:

Objective The effect of different oxygen tensions on the cytoskeleton remodeling of goat temporomandibular joint (TMJ) disc cells were investigated. Methods Goat TMJ disc cells were cultured under normoxia (21% O₂) and hypoxia (2%, 4%, and 8% O₂). Toluidine blue, picrosirius red, and type Ⅰ collagen immunocytochemical staining were performed to observe the changes in cell phenotype under different oxygen levels. Immunofluorescent staining and real-time reverse trans-cription-polymerase chain reaction analysis were then performed to identify actin, tubulin, and vimentin in the cultured disc cells. Results TMJ disc cells still displayed fibroblast characteristics under different oxygen levels and their cytoskeletons had regular arrangement. The fluorescence intensities of actin and vimentin were lowest at 4% O₂ (P<0.05), whereas that of tubulin was highest at 2% O₂ (P<0.05). No significant difference among the other groups was observed (P>0.05). Actin mRNA levels were considerably decreased at 2% O₂ and 4% O₂ in hypoxic conditions, while actin mRNA expression was highest in 21% O₂. Tubulin mRNA levels considerably increased at 2% O₂, while tubulin mRNA expression was lowest in 8% O₂ (P<0.05). Vimentin mRNA expression was lowest at 4% O₂ and highest at 21% O₂, and significant differences were observed between vimentin mRNA expression levels among these oxygen levels (P<0.05). Conclusion Cytoskeletons were recon-structed in different oxygen tensions, and 2% O₂ may be the optimal oxygen level required to proliferate TMJ disc cells.

Key words: temporomandibular joint disc, hypoxia, cytoskeleton, actin, tubulin, vimentin

CLC Number:

Xiao-lan He, Guangjie Bao, Linglu Sun, Xue Zhang, Shanying Bao, Hong Kang. Effect of different oxygen tension on the cytoskeleton remodeling of goat temporomandibular joint disc cells[J]. West China Journal of Stomatology, 2017, 35(4): 362-367.

Figures/Tables 5

Tab 1

Fig 1

Fig 2

Fig 3

Tab 2

References 26

[1]	Stanković S, Vlajković S, Bošković M, et al.Morphological and biomechanical features of the temporomandibular joint disc: an overview of recent findings[J]. Arch Oral Biol, 2013, 58(10):1475-1482.
[2]	Piette E.Anatomy of the human temporomandibular joint. An updated comprehensive review[J]. Acta Stomatol Belg, 1993, 90(2):103-127.
[3]	Benjamin M, Ralphs JR.Biology of fibrocartilage cells[J]. Int Rev Cytol, 2004, 233:1-45.
[4]	Alonso LG, Smith RL, Junior JAF, et al.Ultrastructural evidence of vascularization of the central zone of the tem-poromandibular joint disc in human fetus[J]. Eur J Anat, 2016, 10(2):45-48.
[5]	Yamaguchi A, Tojyo I, Yoshida H, et al.Role of hypoxia and interleukin-1beta in gene expressions of matrix metallo-proteinases in temporomandibular joint disc cells[J]. Arch Oral Biol, 2005, 50(1):81-87.
[6]	Chisnoiu AM, Picos AM, Popa S, et al.Factors involved in the etiology of temporomandibular disorders—a literature review[J]. Clujul Med, 2015, 88(4):473-478.
[7]	DuRaine GD, Brown WE, Hu JC, et al. Emergence of scaffold-free approaches for tissue engineering musculoskeletal car-tilages[J]. Ann Biomed Eng, 2015, 43(3):543-554.
[8]	Shu W, Liu L, Bao G, et al.Tissue engineering of the tem-poromandibular joint disc: current status and future trends[J]. Int J Artif Organs, 2015, 38(2):55-68.
[9]	Hsieh CH, Lin YH, Lin S, et al.Surface ultrastructure and mechanical property of human chondrocyte revealed by atomic force microscopy[J]. Osteoarthr Cartil, 2008, 16(4):480-488.
[10]	Lee JK, Hu JC, Yamada S, et al.Initiation of chondrocyte self-assembly requires an intact cytoskeletal network[J]. Tissue Eng Part A, 2016, 22(3/4):318-325.
[11]	Kuo J, Shi C, Cisewski S, et al.Regional cell density dis-tribution and oxygen consumption rates in porcine TMJ discs: an explant study[J]. Osteoarthr Cartil, 2011, 19(7):911-918.
[12]	Cisewski SE, Zhang L, Kuo J, et al.The effects of oxygen level and glucose concentration on the metabolism of por-cine TMJ disc cells[J]. Osteoarthr Cartil, 2015, 23(10):1790-1796.
[13]	Cernanec J, Guilak F, Weinberg JB, et al.Influence of hy-poxia and reoxygenation on cytokine-induced production of proinflammatory mediators in articular cartilage[J]. Arth-ritis Rheum, 2002, 46(4):968-975.
[14]	Blain EJ.Involvement of the cytoskeletal elements in articu-lar cartilage homeostasis and pathology[J]. Int J Exp Pathol, 2009, 90(1):1-15.
[15]	Benjamin M, Archer CW, Ralphs JR.Cytoskeleton of car-tilage cells[J]. Microsc Res Tech, 1994, 28(5):372-377.
[16]	Chen C, Yin L, Song X, et al.Effects of vimentin disruption on the mechanoresponses of articular chondrocyte[J]. Bio-chem Biophys Res Commun, 2016, 469(1):132-137.
[17]	Jin H, Liang Q, Chen T, et al.Resveratrol protects chondro-cytes from apoptosis via altering the ultrastructural and bio-mechanical properties: an AFM study[J]. PLoS ONE, 2014, 9(3):e91611.
[18]	Leonardi R, Villari L, Piacentini C, et al.Immunolocaliza-tion of vimentin and alpha-smooth muscle actin in dysfunc-tional human temporomandibular joint disc samples[J]. J Oral Rehabil, 2002, 29(3):282-286.
[19]	Thyberg J, Moskalewski S.Role of microtubules in the orga-nization of the Golgi complex[J]. Exp Cell Res, 1999, 246
	(2):263-279.
[20]	郭恒, 段王平, 李琦, 等. 微管蛋白破坏对软骨细胞代谢功能的影响[J]. 中华医学杂志, 2011, 91(15):1036-1040.
	Guo H, Duan WP, Li Q, et al.Disassembly of the tubulin cytoskeleton disrupts articular cartilage chondrocyte homeo-stasis[J]. Natl Med J China, 2011, 91(15):1036-1040.
[21]	Yoshida H, Fukumura Y, Nishida M, et al.The immunohis-tochemical distribution of vimentin in human temporoman-dibular joint samples[J]. J Oral Rehabil, 2004, 31(1):47-51.
[22]	Trickey WR, Vail TP, Guilak F.The role of the cytoskeleton in the viscoelastic properties of human articular chondrocytes[J]. J Orthop Res, 2004, 22(1):131-139.
[23]	Noriega S, Hasanova G, Subramanian A.The effect of ultra-sound stimulation on the cytoskeletal organization of chon-drocytes seeded in three-dimensional matrices[J]. Cells Tis-sues Organs, 2013, 197(1):14-26.
[24]	Pascarelli NA, Collodel G, Moretti E, et al.Changes in ul-trastructure and cytoskeletal aspects of human normal and osteoarthritic chondrocytes exposed to interleukin-1β and cyclical hydrostatic pressure[J]. Int J Mol Sci, 2015, 16(11):26019-26034.
[25]	Magara J, Nozawa-Inoue K, Suzuki A, et al.Alterations in intermediate filaments expression in disc cells from the rat temporomandibular joint following exposure to continuous compressive force[J]. J Anat, 2012, 220(6):612-621.
[26]	孔楠楠, 张文霞, 康宏, 等. 山羊颞下颌关节盘细胞肌动蛋白骨架的研究[J]. 口腔医学研究, 2015, 31(10):953-956.
	Kong NN, Zhang WX, Kang H, et al.Study on the cytoske-leton of cultured goat temporomandibular joint disc cells[J]. J Oral Sci Res, 2015, 31(10):953-956.

目的基因	5’—3’	熔解温度/℃	产物长度/bp
GAPDH	F：CAAGTTCCACGGCACAGTCA	59.9	248
	R：GGTTCACGCCCATCACAAA	59.6
肌动蛋白	F：ATCAAGATTATCGCTCCTCCC	57.7	164
	R：TGCTCGCAGTCCGTTTAGA	57.5
微管蛋白	F：CCCAGGGCAGTGTTTGTAGA	58.3	134
	R：TGACCTCGGGCATAGTTGTT	57.9
波形蛋白	F：ATGACCGCTTCGCCAACTA	59.0	136
	R：TCTCGCATCTCCTCCTCGTA	58.1

目标基因	氧体积分数/%				F值	P值
目标基因	21	8	4	2	F值	P值
肌动蛋白	1	0.510±0.095^ac	0.427±0.402^ab	0.418±0.191^a	7.925	0.009
微管蛋白	1	0.746±0.069^ad	0.980±0.164^d	1.247±0.070^abc	39.600	0.000
波形蛋白	1	0.635±0.099^ac	0.567±0.157^abd	0.625±0.025^ac	19.362	0.010