In Vitro Expansion of Human Nasoseptal Chondrocytes Reveals Distinct Expression Profiles of G 1 Cell Cycle Inhibitors for Replicative,Quiescent,and Senescent Culture Stages

Abstract

In vitro expansion of chondrocytes for tissue-engineering applications is limited by forms of growth arrest known as quiescence and replicative senescence. At the molecular level cyclin-dependent kinase inhibitors (CDKIs) are involved in mediating growth arrest in the G₁ phase of the cell cycle. Using ribonuclease protection assays and immunocytochemical staining methods, we quantitatively analyzed expression profiles of G₁ cell cycle inhibitors at the mRNA and protein levels. These inhibitors included the CDKIs of the CIP/KIP familiy (p21 ^CIP1 , p27 ^KIP1 , and p57 ^KIP2 ) and the INK4 family (p15 ^INK4b , p16 ^INK4a , p18 ^INK4c , and p19 ^INK4d ) as well as the retinoblastoma protein-family (pRb, p107, and p130) and the tumor suppressor p53. Analysis was carried out in proliferating, quiescent, and senescent states of primary cultures of adult human nasoseptal chondrocytes. The most pronounced effect (p < 0.0001) between cultures in proliferation and cultures in growth arrest was an increased expression of the CDKIs p57 ^KIP2 and p15 ^INK4b for quiescent growth arrest, and of p16 ^INK4a , p15 ^INK4b , and p57 ^KIP2 for senescent growth arrest. Thus, these cell cycle inhibitors represent potential candidates for selective intervention to promote cellular multiplication of chondrocytes undergoing in vitro expansion for tissue-engineering applications. Possible methods of modulation include the targeted elimination of specifically identified cell cycle inhibitors by antisense technologies.