Isolation of Endogenous TGF-β1 from Root Canals for Pulp Tissue Engineering: A Translational Study

Abstract

Cell homing for dental pulp tissue engineering has been advocated as a feasible approach to regenerate dental pulp in a clinical setting. In order to develop a translational protocol for clinical application, we wanted to determine the effects of disinfectants on the availability of growth factors from the root canal, the amount that can be obtained in this context, and whether they can be processed for use in tissue engineering procedures. The extraction of growth factors should also be confirmed in a clinical setting. Root canals were prepared in 36 extracted mature teeth, and the amount of TGF-β1 in solution was quantified after different irrigation protocols (sodium hypochlorite, chlorhexidine) and after intracanal medication (calcium hydroxide). Centrifugal filters with a cut-off of 10,000 Da and 3000 Da were used for efficient concentration, and volumes and amounts of retained TGF-β1 were measured at different time points. During conventional endodontic treatment, ethylenediaminotetraacetic acid (EDTA) solution was collected after ultrasonic activation from the root canals of mature teeth of 38 patients, and growth factor content was quantified via enzyme-linked immunosorbent assay (ELISA). Irrigation with sodium hypochlorite reduced TGF-β1 release into EDTA. This effect was partially reversed by canal enlargement after the use of sodium hypochlorite and by subsequent use of calcium hydroxide. A few minutes of centrifugation with a cut-off of 10,000 Da reduced the initial volume of the irrigant by 90% and led to a continuous increase in concentration to the same extent. Furthermore, TGF-β1 was obtained from root canals of mature teeth during endodontic treatment in quantities that have been shown to elicit desirable cellular responses in a subsequent clinical application. A mixture with a suitable scaffold material and injection into the root canal has the potential to promote dental pulp regeneration.

Keywords: cell homing; dental pulp; dentin matrix proteins; endodontics; regeneration; tissue engineering.

Figure 1

Envisioned clinical procedure. After root canal preparation and disinfection, EDTA is used to remove the smear layer. After refreshment of EDTA, the solution is activated by ultrasound, which releases growth and differentiation factors from the dentin surface. The solution is removed from the canal, and proteins are concentrated via a chairside centrifugation step. A hydrogel biomaterial, e.g., a fibrin-based scaffold, is mixed with concentrated dentin matrix proteins and injected back into the root canal in contact with the periapical tissues.

Figure 2

Collection of growth factors from root canals in an ex vivo model. Release of TGF-β1 depending on irrigant solution after a 3-step-protocol including initial preparation, root canal enlargement, and the use of an intracanal dressing. (A) Concentration and (B) absolute mass of soluble TGF-β1 in EDTA collected from root canals after ultrasonic activation (n = 12). (C) Retrieved volumes of EDTA and number of collections necessary to obtain a minimum total volume of 100 µL (n = 36). Depicted are median values and 25–75% percentiles (column and error bars) as well as single measurements (points). Width of distribution of points proportionate to the number of points. Asterisks indicate statistical differences between groups.

Figure 3

Volume of EDTA and concentration of TGF-β1 after centrifugation in filters with a cut-off at 3000 or 10,000 Da. Both parameters were measured at 6 time points (n = 6). Depicted are medians and 25–75% percentiles.

Figure 4

Release of TGF-β1 into EDTA and saline for all 38 teeth (A) and according to tooth type (B), clinical situation (C), patient age (D) and extent of the coronal restoration (E). Depicted are median values with 25–75% percentiles. Asterisks indicate statistical differences between groups.