Thermosensitive Chitosan–Gelatin–Glycerol Phosphate Hydrogels as a Cell Carrier for Nucleus Pulposus Regeneration: An In Vitro Study Yung-Hsin Cheng, Ph.D., 1 Shu-Hua Yang, M.D., Ph.D., 2 Wen-Yu Su, Ph.D., 1 Yu-Chun Chen, Ph.D., 1 Kai-Chiang Yang, Ph.D., 1 Winston Teng-Kuei Cheng, Ph.D., 3–5 Shinn-Chih Wu, Ph.D., 3 and Feng-Huei Lin, Ph.D. 1,6 Injectable hydrogel is one of the great interests for tissue engineering and cell encapsulation. In the study, the gelatin molecules were added to the thermosensitive chitosan=b-glycerol phosphate (C=GP) disodium salt hy- drogels to form chitosan=gelatin=b-glycerol phosphate (C=G=GP) disodium salt hydrogels which were applied as a cell carrier for nucleus pulposus (NP) regeneration. The gelation temperature, gelation time, and gel strength of the C=G=GP hydrogels were analyzed by the rheometer. NP cells were then harvested from the intervertebral discs of the adult New Zealand white rabbits and cultured in monolayer or in C=G=GP hydrogel, respectively. The cell viability, material-mediated cytotoxicity, cell proliferation, production of sulfated glycosaminoglycans, anabolic=catabolic gene expressions, and extracellular matrix-related gene expressions of the NP cells were demonstrated. The results show that the sol=gel transition temperature of the C=G=GP hydrogel was in the range of 31.1–33.88C at neutral pH value, the gelation time was shortened, and the gel strength also improved at body temperature when compared with the C=GP hydrogel. Among those, C=GP with 1% gelatin addition showed the most promising gelation time and gel strength and were utilized in the later experiments. From the results of cell activity, cytotoxicity, and cell proliferation assays, NP cells cultured in C=G=GP hydrogel had normal cell viability and cell proliferation that indicated the hydrogel was noncytotoxicity. The amounts of sulfated glycosaminoglycans of NP cells cultured in C=G=GP hydrogels were significantly higher than mono- layer cultured. Considering the extracellular matrix-related gene expression, type II collagen and aggrecan of NP cells cultured in the hydrogels greatly increased than those in monolayer culture. On the contrary, the unfa- vorable gene expression, such as that of type I collagen, was decreased significantly. The results reveal that gelatin added into C=GP hydrogel significantly shortened the gelation time and improved the gel strength without influencing the biocompatibility. NP cells cultured in the C=G=GP hydrogel also displayed better gene expressions when compared with the monolayer culture. This study indicates that using chitosan=gelatin hy- drogel for NP cell culture is feasible and may apply in minimal invasive intervertebral disc surgery in the future. Introduction T he intervertebral disc (IVD) is composed of central nucleus pulposus (NP), outer annulus fibrosus, and upper and lower cartilage endplates (CEPs). IVD plays an important role by providing adequate mechanical strength in response to external stress, thus protecting the spine and brain from damage. The major components of NP are disc- matrix proteins, proteoglycans, and collagen which have gelatinous structure and thus they give resilience after shock absorption. 1 With disc degeneration, proteoglycans of the NP are progressively lost, leading to poor hydrodynamic transfer. Simultaneously, the integrity of the annulus fi- brosus is degraded and radial fissures are generated. 2 The CEPs are also affected by the degenerative process accom- panying with ossification. As known, the NP is an avascular tissue in the body; the nutrient supply depends on the capillaries of the surrounding tissue, and it diffuses through 1 Institute of Biomedical Engineering, College of Engineering and College of Medicine, National Taiwan University, Taipei, Taiwan. 2 Department of Orthopedics, National Taiwan University Hospital, Yun-Lin Branch, Yun-Lin, Taiwan. 3 Department of Animal Science and Technology, National Taiwan University, Taipei, Taiwan. 4 Department of Animal Science and Biotechnology, Tunghai University, Taichung, Taiwan. 5 Institute of Biotechnology, National Taiwan University, Taipei, Taiwan. 6 Division of Medical Engineering, National Health Research Institute, Miaoli County, Taiwan. TISSUE ENGINEERING: Part A Volume 16, Number 2, 2010 ª Mary Ann Liebert, Inc. DOI: 10.1089=ten.tea.2009.0229 695 Downloaded by 18.206.13.133 from www.liebertpub.com at 05/21/20. For personal use only.