title: Establishment of a promising human nucleus pulposus cell line for intervertebral disc tissue engineering.
abstract: Low-back pain caused by intervertebral disc degeneration could be recovered by the regeneration of the nucleus pulposus (NP). This study aimed to establish a chondrogenic recovery model with promising a human NP (hNP) cell line, an immortalized hNP (ihNP), which could be a screening platform to identify regenerative drugs. The ihNP cells were created from primary human NP cells transfected with a retroviral vector-driven HPV16 E6/E7. Growth properties and characteristics of ihNP were evaluated by comparing with parental NP cells. Successful immortalization of ihNP cells stably expressed HPV 16 E6/E7 mRNA. The doubling time of ihNP was shortened to 53.16±2.63 h compared with parental hNP-P1. Cell cycle regulators, including p53, p21, and pRB were downregulated compared to parental hNP-P1. The in vivo neoplastic forming assay also demonstrated that the ihNP was nontumorigenic. After 25 generations of cell cultures, the ihNP cells, yet stably expressed chondrogenic genes, including (SOX9), type II collagen (Col II), aggrecan, decorin, biglycan, and versican. Higher expressions of chondrogenic proteins, including Col II, phosphorylated SOX9 (p-SOX9), and CD44 were also determined. Under the stressful inflammatory conditions induced by lipopolysaccharides (LPS), the regenerative and anti-inflammatory potentials of ihNP in two-dimensional culture with the presence of platelet-rich plasma (PRP) were evaluated by reverse transcriptase polymerase chain reaction. PRP showed significant effects on restoring diminished chondrogenic markers and deleterious inflammatory responses induced by LPS in ihNP. The therapeutic potentials of ihNP in three-dimensional neocartilage model could also be exerted by PRP using histological evaluation and immunological staining. Hence, the established ihNP cells can provide a chondrogenic recovery model as a regenerative drug screening tool for further regenerative drug discovery and development.
authors.affiliation: 1 College of Oral Medicine, Taipei Medical University , Taipei, Taiwan, Republic of China .
authors.forename: Ming-Che
authors.initials: MC
authors.lastname: Liu
authors.forename: Wei-Hong
authors.initials: WH
authors.lastname: Chen
authors.forename: Ling-Chiao
authors.initials: LC
authors.lastname: Wu
authors.forename: Wei-Che
authors.initials: WC
authors.lastname: Hsu
authors.forename: Wen-Cheng
authors.initials: WC
authors.lastname: Lo
authors.forename: Shauh-Der
authors.initials: SD
authors.lastname: Yeh
authors.forename: Ming-Fu
authors.initials: MF
authors.lastname: Wang
authors.forename: Rong
authors.initials: R
authors.lastname: Zeng
authors.forename: Win-Ping
authors.initials: WP
authors.lastname: Deng
mesh_terms: D000328:Adult; D000818:Animals; D063646:Carcinogenesis / Q000378:metabolism / Q000473:pathology; D018929:Cell Culture Techniques / Q000379:methods*; D002453:Cell Cycle; D002461:Cell Line, Transformed; D049109:Cell Proliferation; D048430:Cell Shape; D016922:Cellular Senescence; D020219:Chondrogenesis / Q000235:genetics; D020869:Gene Expression Profiling; D006801:Humans; D007403:Intervertebral Disc / Q000166:cytology* / Q000502:physiology; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D008954:Models, Biological; D012038:Regeneration; D023822:Tissue Engineering / Q000379:methods*