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Acta Phys. -Chim. Sin.  2017, Vol. 33 Issue (12): 2446-2453    DOI: 10.3866/PKU.WHXB201706133
ARTICLE     
Raman Spectroscopic Analysis of Chondrocyte Dedifferentiation during in vitro Proliferation
Lu-Di JIN1,Jing-Jing XU2,Yong ZHANG3,Yue-Zhou YU2,Chang LIU3,Dong-Ping ZHAO3,An-Pei YE1,3,*()
1 Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, P. R. China
2 Peking-Tsinghua Center for Life Sciences, Beijing 100871, P. R. China
3 Key Laboratory for the Physics & Chemistry of Nano-devices, School of Electronics Engineering & Computer Science, Peking University, Beijing 100871, P. R. China
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Abstract  

Seeded chondrocytes play a crucial role in current cartilage tissue engineering, yet both the quality and quantity of these cells could be impaired owing to cell dedifferentiation during in vitro proliferation. Here, we used micro-Raman spectroscopy to investigate changes in cellular components upon monolayer culturing of primary rat chondrocytes through multiple passages. Based on the average spectral profiles, we detected a series of Raman peaks and recognized related radicals such as nucleobases, pyranose rings, sulfate, tyrosine, proline, and amides at the single-chondrocyte level. Quantitative analysis of the Raman peak intensities showed that nucleic acids (at 789, 1094, 1576 cm-1) decreased significantly from passage 1 (P1) to passage 4 (P4), whereas lipids (at 1304 cm-1) and phosphate (at 957 cm-1) increased significantly. Moreover, the syntheses of two major hyaline cartilage-associated proteins, aggrecan and type-2 collagen, were impeded, as indicated by the marked decline in the levels of their specific components (glycosaminoglycan at 1042, 1063, 1126, 1160 cm-1, and hydroxyproline at 1207 cm-1). Taken together, these features reveal the diminished propagation and secretion abilities of passaged chondrocytes needed for matrix-induced implantation, and shed light on the molecular mechanism of chondrocyte dedifferentiation.



Key wordsChondrocyte      Dedifferentiation      Single-cell analysis      Micro-Raman spectroscopy      Molecular mechanism     
Received: 02 May 2017      Published: 13 June 2017
MSC2000:  O641  
Fund:  the National Natural Science Foundation of China(U1636110);the National Key Technologies R & D Program of China(2012BAF14B14);the Medicine-Informatics Interdisciplinary Project of Peking University, China(2014-MI-19)
Corresponding Authors: An-Pei YE     E-mail: yap@pku.edu.cn
Cite this article:

Lu-Di JIN,Jing-Jing XU,Yong ZHANG,Yue-Zhou YU,Chang LIU,Dong-Ping ZHAO,An-Pei YE. Raman Spectroscopic Analysis of Chondrocyte Dedifferentiation during in vitro Proliferation. Acta Phys. -Chim. Sin., 2017, 33(12): 2446-2453.

URL:

http://www.whxb.pku.edu.cn/10.3866/PKU.WHXB201706133     OR     http://www.whxb.pku.edu.cn/Y2017/V33/I12/2446

Fig 1 Configuration of the experimental confocal Raman spectrometry platform Color online
Fig 2 Normalized Raman spectra of P1-P4 cells The colored (P1 = orange, P2 = violet, P3 = brown, P4 = green) solid lines indicate the average spectra normalized according to the peak intensities at 1001 cm-1. The grey-shaded areas represent the standard errors. The Raman excitation wavelength is 532 nm. Color online
Fig 3 Common peak selections from the average Raman spectra of P1-P4 chondrocytes The colored (P1 = orange, P2 = violet, P3 = brown, P4 = green) solid lines indicate the normalized spectra, which are vertically offset for clarity. The Raman excitation wavelength is 532 nm. Color online
Raman peak wavenumbers/cm-1Wavenumbers in references/cm-1Peak assignments
P1P2P3P4
789787791789787DNA/RNA (O-P-O stretch, cytosine, uracil, thymine)
861860860857858Proline
935933942942937Ring breathing mode of tyrosine, and C-C stretch of proline ring
954-955954957Phosphate (hydroxyapatite)
10011000100110001001Symmetric ring breathing mode of phenylalanine
10441044104410441042Pyranose ring
106110591063-1063Sulfate
10881091109310911094DNA/RNA (O-P-O stretch)
11211123112311261126Pyranose ring
11631161116111631160Pyranose ring
12071204120512021207Aromatic ring stretch
12451247124412451245Amide Ⅲ (CH2 wagging, C-N stretch)
1303-130613041304Lipids (CH2/CH3 twisting)
14421439144414441446Lipids/proteins (CH2 bending, amide Ⅰ)
--1515-1515C=C stretch of carotenoid
15721574157615771576Guanine-N3
16521654165516561656Amide Ⅰ (C=O stretch)
Table 1 Raman peak wavenumbers of P1-P4 chondrocytes and respective vibrational assignments24-35
Fig 4 Comparisons of relative Raman peak intensities from P1–P4 chondrocytes (p < 0.05) Dependences of the peak intensities are shown as functions of chondrocyte passage number (1-4): (a) 787, 1094, 1576 cm-1; (b) 1042, 1063, 1126, 1160 cm-1; (c) 1207 cm-1; (d) 1246 cm-1 to 1270 cm-1; (e) 1304 cm-1; (f) 957 cm-1.Data represent the normalized mean intensities of Raman peaks, and bars represent the standard errors. Color online
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