Paracrine Effects of Mesenchymal Stem Cells on Dental Tissues - in vitro and in vivo studies

Abstract

In regenerative medicine or dentistry, it has been reported that stem cells induce the regenerative potential of injured tissues. In the present thesis, pulp and periapical tissues as well as pulpal cells were exposed to bioactive soluble molecules secreted by bone marrow mesenchymal stem cells (BMSC) cultured in vitro to determine the paracrine effects of MSC on tissue healing and regeneration. In Study I, the proliferation and osteo/odontogenic differentiation of human dental pulp cells (hDPC) exposed in vitro to the exogenous recombinant growth differentiation factor-5 (GDF-5) and to a cocktail of soluble growth factors released by bone marrow stem cells in a conditioned culture medium (CM) were evaluated. Cell proliferation was examined by MTT, and osteo/odontogenic differentiation was assessed by Real-Time Quantitative Reverse Transcription PCR, alkaline phosphatase (ALP) staining, osteocalcin (OC) immunoreactivity and Alizarin Red Staining. It was found that CM collected from cultures of BMSC has higher osteo/odontogenic inductive effect on hDPC than GDF-5. Study II was designed to evaluate the influence of CM on the healing responses of the dental pulp and periodontium of rat molars, following immediate replantation. CM had no effect on vascular endothelial growth factor (VEGF) mRNA and ALP mRNA in the dental pulp after 3 days, whereas an up-regulation of ALP mRNA was found in the tooth socket of the replanted teeth. Observations after 90 days showed that CM reduced the occurrence of external cervical and surface resorption and prevented extensive dentin production in replanted teeth. Following the disclosure in Study II that CM had a protective effect on the pulp tissue following replantation, Study III was undertaken in order to investigate the underlying effect of CM on the release of inflammatory cytokines from hDPC in vitro, and on the gene expression of inflammatory cytokines following tooth replantation. In vitro, CM significantly stimulated production of prostaglandin E2 (PGE2), the inflammatory cytokines interleukin IL-10, -6 and -8, and chemokine RANTES, in hDPC compared with the control cells. Three days after tooth replantation, significantly lower mRNA levels of IL-1β, and-6, and TNF-α were associated with CM than with untreated replanted teeth. These studies showed that BMSC-CM stimulates early differentiation and matrix mineralization, and the expression of inflammatory mediators in hDPC in vitro. BMSC-CM seems to attenuate the initial inflammatory reaction in pulp tissue, and enhance pulpal and periodontal healing following replantation of rat molars.