The potential application of concentrated growth factor in pulp regeneration: an in vitro and in vivo study

Concentrated growth factor (CGF), as a natural biomaterial, is known to contain platelets, cytokines, and growth factors to facilitate the healing process, but there has been little information acquired in regenerative endodontics. The purpose of this study was to investigate the effects of CGF on proliferation, migration, and differentiation in human dental stem pulp cells (hDPSCs) exposed to lipopolysaccharide (LPS) in vitro and its potential role in pulp regeneration of the immature teeth in vivo.

In vitro experiments: CGF-conditioned medium were extracted by freeze-dried method. hDPSCs were isolated and identified. The proliferative potential of hDPSCs with different concentration of CGF and LPS was evaluated by Cell Counting Kit-8. Migration capacity was analyzed by Transwell assays, odonto/osteoblastic differentiation was determined by measuring alkaline phosphatase (ALP) activity using ALP staining, and the extent of mineralization was evaluated by using Alizarin red S staining. The mRNA expression level of DMP-1, DSPP, OPN, Runx2, and OCN were determined by quantitative polymerase chain reaction (qPCR). In vivo experiments: CGF were used as root canal filling agent of the immature single-rooted teeth in the beagle dogs. The teeth were then radiographed, extracted, fixed, demineralized, and subjected to histologic analyses at 8 weeks. The newly formed dentine-pulp complex and the development of apical foramen were evaluated by the hematoxylin-eosin (HE) and Masson trichrome technique. Soft tissues were analyzed by immunohistochemical staining of vascular endothelial growth factor (VEGF) and Nestin.

In vitro experiments: The cultured cells exhibited the characteristics of mesenchymal stem cell. The treatment of LPS significantly increased the expression of TNF-α, IL-1β, IL-6, and IL-8 in hDPSCs, and CGF inhibited the mRNA expression of IL-8 in LPS-stimulated hDPSCs. The proliferation values of the CGF group in LPS-stimulated hDPSCs were significantly higher than that of the control group from day 3 to day 7 (P < 0.05). In addition, the number of migratory cells of the CGF group was greater than that of the control group at 24 h with or without LPS treatment. ALP activities increased gradually in both groups from day 4 to day 7. The mineralized nodules and the expression of odontogenesis-related genes DMP-1 and DSPP, osteogenesis-related genes OPN, Runx2, and OCN were dramatically enhanced by CGF in LPS-stimulated hDPSCs at days 21 and 28. In vivo experiments: In CGF treated group, the results of radiograph, HE, and Masson trichrome staining showed a continuing developed tooth of the immature teeth in the beagle dogs (i.e., the ingrowth of soft tissues into the root canal, the thickened internal root dentin walls, and the closed apex), which resembled the normal tooth development in the positive control group. The immunohistochemical staining showed that VEGF and Nestin were both moderately expressed in the regenerated pulp-like tissues which indicating the vascularization and innervation.

CGF has a positive effect on the proliferation, migration, and differentiation of hDPSCs exposed to LPS in vitro, and it can also promote the regeneration of dentine-pulp complex of the immature teeth in the beagle dogs in vivo. Therefore, CGF could be a promising alternative biomaterial in regenerative endodontics.