Tooth slice/scaffold model of dental pulp tissue engineering

Abstract

Multipotency is a defining characteristic of post-natal stem cells. The human dental pulp contains a small subpopulation of stem cells that exhibit multipotency, as demonstrated by their ability to differentiate into odontoblasts, neural cells, and vascular endothelial cells. These discoveries highlight the fundamental role of stem cells in the biology of the dental pulp and suggest that these cells are uniquely suited for dental pulp tissue-engineering purposes. The availability of experimental approaches specifically designed for studies of the differentiation potential of dental pulp stem cells has played an important role in these discoveries. The objective of this review is to describe the development and characterization of the Tooth Slice/Scaffold Model of Dental Pulp Tissue Engineering. In addition, we discuss the multipotency of dental pulp stem cells, focusing on the differentiation of these cells into functional odontoblasts and into vascular endothelial cells.

Figure 1.

Tooth Slice/Scaffold Model of Dental Pulp Tissue Engineering. (A) A 1-mm-thick tooth slice is cut from the cervical region of a non-carious human third molar. (B) Tooth slice showing an empty pulp cavity after removal of the dental pulp. (C) A highly porous PLLA scaffold is cast within the pulp chamber. (D) Implantation of a tooth slice/scaffold seeded with dental pulp stem cells in the subcutaneous space of the dorsum of an immunodeficient mouse. (E) Bilateral tooth slice/scaffolds at the termination of the experimental period (i.e., 3 wks after transplantation), showing a highly vascularized tissue in the pulp chamber.

Figure 2.

Dental pulp stem cells differentiate into dentinogenic cells. (A,B) Representative photomicrographs of dental pulps engineered with stem cells from human exfoliated primary teeth (SHED) seeded in a tooth slice/scaffold and transplanted into an immunodeficient mouse. Hematoxylin and eosin staining at 200x (A) and 400x (B). (C) Cells aligned close to the pre-dentin showed strong staining for human DSP (brown) at 400x. (D) Photomicrographs of confocal microscopy performed in tooth slices retrieved from immunodeficient mice 32 days after transplantation. The experimental conditions were as follows: dental pulp (tooth slice of a freshly extracted non-carious human molar from which the pulp was not removed); tooth slice/scaffold + SHED (tooth slice/scaffold seeded with SHED); tooth slice/scaffold (tooth slice/scaffold without SHED); and tooth slice (tooth slice without scaffold and without SHED). All mice received 4 to 5 intraperitoneal injections of 41.6 nmol/g of body weight of tetracycline hydrochloride.

Figure 3.

Dental pulp stem cells differentiate into vasculogenic cells. (A) Photomicrograph of SHED cells cultured in 3-D collagen type I matrices and stimulated with rhVEGF₁₆₅. (B) Confocal microscopy of SHED cells cultured in 3-D matrices and stimulated with rhVEGF₁₆₅. (C) Immunohistochemistry for human Factor VIII, a histological marker for identification of blood vessels at 200x. Arrows point to Factor VIII-positive (red) blood vessels. (D) SHED stably transduced with LacZ were seeded in tooth slice/scaffolds and implanted into immunodeficient mice. After 21 days, tooth slice/scaffolds were retrieved, demineralized, and stained with x-gal. Arrows point to β-galactosidase-positive blood vessels (blue) in the engineered tissue, confirming that the SHED cells differentiated into endothelial cells in vivo. Representative photomicrograph of hematoxylin-and-eosin-stained tissue section at 400x. (E) Blood vessel sectioned longitudinally within a dental pulp engineered within a tooth slice/scaffold seeded with SHED (200x).