Label-Free Characterization of Collagen Crosslinking in Bone-Engineered Materials Using Nonlinear Optical Microscopy

Chao-Wei Hung; Nirmal Mazumder; Dan-Jae Lin; Wei-Liang Chen; Shih-Ting Lin; Ming-Che Chan; Guan-Yu Zhuo

doi:10.1017/S1431927621000295

Label-Free Characterization of Collagen Crosslinking in Bone-Engineered Materials Using Nonlinear Optical Microscopy

Microsc Microanal. 2021 Apr 8:1-11. doi: 10.1017/S1431927621000295. Online ahead of print.

Authors

Chao-Wei Hung¹, Nirmal Mazumder², Dan-Jae Lin³, Wei-Liang Chen⁴, Shih-Ting Lin⁵, Ming-Che Chan^{6

7}, Guan-Yu Zhuo^{5

8}

Affiliations

¹ PhD Program for Biomedical Engineering and Rehabilitation Science, China Medical University, No. 91, Hsueh-Shih Road, Taichung40402, Taiwan R.O.C.
² Department of Biophysics, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, Karnataka576104, India.
³ School of Dentistry, College of Dentistry, China Medical University, No. 91, Hsueh-Shih Road, Taichung40402, Taiwan R.O.C.
⁴ Center for Condensed Matter Sciences, National Taiwan University, Taipei10617, Taiwan R.O.C.
⁵ Integrative Stem Cell Center, China Medical University Hospital, No. 2, Yude Road, Taichung40447, Taiwan R.O.C.
⁶ Institute of Photonic System, College of Photonics, National Chiao-Tung University, Tainan71150, Taiwan R.O.C.
⁷ Institute of Biophotonics, National Yang-Ming University, No. 155, Sec. 2, Linong Street, Beitou District, Taipei City112, Taiwan R.O.C.
⁸ Institute of New Drug Development, China Medical University, No. 91, Hsueh-Shih Road, Taichung40402, Taiwan R.O.C.

PMID: 33829983
DOI: 10.1017/S1431927621000295

Abstract

Engineered biomaterials provide unique functions to overcome the bottlenecks seen in biomedicine. Hence, a technique for rapid and routine tests of collagen is required, in which the test items commonly include molecular weight, crosslinking degree, purity, and sterilization induced structural change. Among them, the crosslinking degree mainly influences collagen properties. In this study, second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy are used in combination to explore the collagen structure at molecular and macromolecular scales. These measured parameters are applied for the classification and quantification among the different collagen scaffolds, which were verified by other conventional methods. It is demonstrated that the crosslinking status can be analyzed from SHG images and presented as the coherency of collagen organization that is correlated with the mechanical properties. Also, the comparative analyses of SHG signal and relative CARS signal of amide III band at 1,240 cm−1 to δCH2 band at 1,450 cm−1 of these samples provide information regarding the variation of the molecular structure during a crosslinking process, thus serving as nonlinear optical signatures to indicate a successful crosslinking.

Keywords: coherent anti-Stokes Raman scattering; collagen crosslinking; molecular orientation; nonlinear optical microscopy; second harmonic generation.