All‐trans retinoic acid and human salivary histatin‐1 promote the spreading and osteogenic activities of pre‐osteoblasts in vitro

Abstract

Cell‐based bone tissue engineering techniques utilize both osteogenic cells and biomedical materials, and have emerged as a promising approach for large‐volume bone repair. The success of such techniques is highly dependent on cell adhesion, spreading, and osteogenic activities. In this study, we investigated the effect of co‐administration of all‐trans retinoic acid (ATRA) and human salivary peptide histatin‐1 (Hst1) on the spreading and osteogenic activities of pre‐osteoblasts on bio‐inert glass surfaces. Pre‐osteoblasts (MC3T3‐E1 cell line) were seeded onto bio‐inert glass slides in the presence and absence of ATRA and Hst1. Cell spreading was scored by measuring surface areas of cellular filopodia and lamellipodia using a point‐counting method. The distribution of fluorogenic Hst1 within osteogenic cells was also analyzed. Furthermore, specific inhibitors of retinoic acid receptors α, β, and γ, such as ER‐50891, LE‐135, and MM‐11253, were added to identify the involvement of these receptors. Cell metabolic activity, DNA content, and alkaline phosphatase (ALP) activity were assessed to monitor their effects on osteogenic activities. Short‐term (2 h) co‐administration of 10 μm ATRA and Hst1 to pre‐osteoblasts resulted in significantly higher spreading of pre‐osteoblasts compared to ATRA or Hst1 alone. ER‐50891 and LE‐135 both nullified these effects of ATRA. Co‐administration of ATRA and Hst1 was associated with significantly higher metabolic activity, DNA content, and ALP activity than either ATRA or Hst1 alone. In conclusion, co‐administration of Hst1 with ATRA additively stimulated the spreading and osteogenicity of pre‐osteoblasts on bio‐inert glass surfaces in vitro.

Keywords

• all‐trans retinoic acid
• cell spreading
• histatin‐1
• osteogenic cells
• pre‐osteoblasts