Ca²⁺ promotes the calcification of induced bone-like tissue in vitro

In recent experiments, bone induction from immature muscular tissue (IMT) in vitro using BMP as a source of cytokine, and expanded-polytetrafluoroethylene (e-PTFE) as a scaffold has been attempted. These conditions, however, could not induce bone. Instead, bone-like tissue was induced. Furthermore, this induced bone-like tissue has the potential to ossify after implantation in vivo. However, the calcification level of such induced bone-like tissue in vitro was low. Objectives: The aim of this study was to promote the calcification of induced bone-like tissue, and examine the influence of Ca²⁺ in this process. Methods: BMP was dissolved in 1M of PBS. IMTs, which were obtained from Sprague-Dawley day 20 embryonic rats, were placed into a homogenizer with 10 ml of dissolved BMP and homogenized. After the homogenized IMT was placed on e-PTFE, and was cultured for 2 weeks in a-MEM supplemented with 15% FBS, 50mg/ml ascorbic acid, 10mM Na-b-glycerophosphate. In addition, 10mM of CaCl₂ was added to the medium. After that, Hematoxylin-Eosin (H-E) staining, von Kossa staining and immunostaining for collagen type I, osteopontin and osteocalcin were performed for histological observation. RT-PCR was performed to quantify the expression of collagen type I, osteopontin and osteocalcin on days 3, 7, 10 and 14. Results: Bone-like tissue, containing osteoblastic cells and osteoids, was observed by H-E staining. Strong calcareous deposition was observed by von Kossa staining. Furthermore, collagen type I, osteopontin and osteocalcin were detected in osteoids by immunostaining. The expression of all osteoblastic marker genes was confirmed on each day and more importantly, the gene expression level of osteocalcin was upregulated more than in the previous experiment. Conclusion: The addition of CaCl₂ in the medium was thus found to promote the calcification of induced bone-like tissue in vitro and it was also considered to be very effective for this model of tissue engineering.