Dental Pulp Tissue Regeneration Using Dental Pulp Stem Cells Isolated and Expanded in Human Serum

Abstract

Introduction: Dental pulp-derived stem cells (DPSCs) have the potential to regenerate dentin and dental pulp tissue because of their differentiation capacity and angiogenic properties. However, for regenerative approaches to gain regulatory and clinical acceptance, protocols are needed to determine more feasible ways to cultivate DPSCs, namely, without the use of xenogeneic-derived components (animal sera) and exogenous growth factors.

Methods: In this study, human DPSCs were isolated from third molars and expanded in standard culture conditions containing fetal bovine serum (DPSCs-FBS) or conditions containing human serum (DPSCs-HS). After cell characterization and evaluation of their angiogenic secretome, DPSCs were seeded in tooth slice/scaffolds and implanted subcutaneously into immunodeficient mice. After 30 days, tooth slices were retrieved and evaluated for dental pulp tissue regeneration. Immunohistochemistry and confocal microscopy were used to quantify blood vessel formation and evaluate predentin and dentin formation.

Results: After culture, DPSCs-HS produced concentrations of angiogenic growth factors equivalent to DPSCs-FBS. Additionally, in DPSCs-HS, several angiogenic factors were produced in at least 1-fold higher concentrations than in DPSCs-FBS. In vivo, it was determined that DPSCs-HS produced a robust angiogenic response and regeneration of dentin equivalent to DPSCs-FBS.

Conclusions: These findings show that DPSCs can be isolated and expanded to clinical scale numbers in media devoid of animal serum or exogenous growth factors and still maintain their pulp regenerative properties. The implications of these findings are significant for further development of clinical protocols using DPSCs in cell therapies.

Keywords: Angiogenesis; cell therapy; dental pulp stem cells; dentin; pulp tissue engineering.

Figure 1

DPSC characterization. (A) Photomicrographs of DPSCs cultured in media containing FBS or HS. (B) Cell proliferation, as measured by population doubling time (PDT), over time between DPSCs expanded in FBS vs. those expanded in HS. (C) Stem cell surface marker expression of passage 5 DPSCs. DPSC populations expanded in media containing FBS or HS consistently yielded high proportions of cells that were positive for the mesenchymal stem cell markers CD73, CD90, and CD105.

Figure 2

Angiogenic factors produced by DPSCs-HS vs. DPSC-FBS. (A) Representative results of anigogenic proteome profiler arrays probed with serum-free conditioned medium (CM) from DPSCs-HS and DPSCs-FBS; boxes indicate individual factors in duplicate. (B) Quantitative analysis of angiogenic factors and chemokines in CM from DPSC-FBS and DPSC-HS differentially expressed by greater than one-fold. * denotes P < 0.05 relative to FBS condition.

Figure 3

DPSC induced angiogenesis in tooth slice. (A) High magnification histological images of blood vessel formation following hematoxylin and eosin (H&E) staining to evaluate total vessel formation and Factor VIII staining to identify human DPSC derived vessels (magnification, 200x; green arrows delineate vessels with brown anti-human Factor VIII staining). (B) Quantitative analysis of total vessel formation in condition following transplantation of DPSC-HS and DPSCS-FBS. (C) Quantitative analysis of human derived vessels following transplantation of DPSC-HS and DPSCS-FBS.

Figure 4

Predentin and dentin formation in tooth slice following transplantation of DPSC-HS and DPSC-FBS. (A) Histologic evaluation shows the cellularity of the tissue lining the predentin of engineered dental pulps in tooth slice/scaffolds seeded with DPSC-HS and DPSC-FBS compared to normal/control dental pulp (magnification, 400x; arrows show predentin region). (B) Representative confocal microscopic photomicrograph showing fluorescent lines (resultant of tetracycline injections) in dental pulp of a mouse incisor, indicating continuous dentin deposition (dentinogenesis) over time. Human tooth slices recovered after 30 days showing the presence of tetracycline lines found either in DPSC-HS or DPSC-FBS groups, indicating the presence of similar dentinogenic activity within engineered pulp tissue of tooth slices (arrows delineate fluorescent lines).