Effect of intermittent PTH(1-34) on human periodontal ligament cells transplanted into immunocompromised mice

Abstract

Residual periodontal ligament (PDL) cells in the damaged tissue are considered a prerequisite for a successful regeneration of the periodontal architecture with all its components, including gingiva, PDL, cementum, and bone. Among other approaches, current concepts in tissue engineering aim at a hormonal support of the regenerative capacity of PDL cells as well as at a supplementation of lost cells for regeneration. Here, we investigated how far an anabolic, intermittent parathyroid hormone (iPTH) administration would enhance the osteoblastic differentiation of PDL cells and the cellular ability to mineralize the extracellular matrix in an in vivo transplantation model. PDL cells were predifferentiated in a standard osteogenic medium for 3 weeks before subcutaneous transplantation into CD-1 nude mice using gelatin sponges as carrier. Daily injections of 40 μg/kg body weight PTH(1-34) or an equivalent dose of vehicle for 4 weeks were followed by explantation of the specimens and an immunohistochemical analysis of the osteoblastic marker proteins alkaline phosphatase (ALP), osteopontin, and osteocalcin. Signs of biomineralization were visualized by means of alizarin red staining. For verification of the systemic effect of iPTH application, blood serum levels of osteocalcin were determined. The osteogenic medium stimulated the expression of ALP and PTH1-receptor mRNA in the cultures. After transplantation, iPTH resulted in an increased cytoplasmic and extracellular immunoreactivity for all markers investigated. In contrast to only sporadic areas of mineralization under control conditions, several foci of mineralization were observed in the iPTH group. Blood serum levels of osteocalcin were elevated significantly with iPTH. These data indicate that the osteoblastic differentiation of human PDL cells and their ability for biomineralization can be positively influenced by iPTH in vivo. These findings hold out a promising prospect for the support of periodontal regeneration.

Figures

FIG. 1.

Induction of enhanced osteoblastic differentiation of periodontal ligament (PDL) cells by dexamethasone. After 3 weeks of culture, the alkaline phosphatase (ALP) activity and parathyroid hormone 1-receptor (PTH1-R) transcript expression increased significantly compared to the vehicle-treated controls. The bar graph shows the mean±SEM of six independent cultures that were assayed in duplicate. **p<0.01, dexamethasone-treated group versus vehicle control; ***p<0.001, dexamethasone-treated group versus. vehicle control.

FIG. 2.

PTH(1–34)-induced increase of osteocalcin serum levels in vivo as determined by ELISA after 4 weeks. The bar graph shows the mean±SEM of six mice per group. **p<0.05, PTH(1–34) versus vehicle control.

FIG. 3.

Proof of human cells in the specimens explanted from six CD-1® nude mice. Specimens were processed for immunohistochemical detection of human cell nuclei to identify human PDL cells (left panel; open arrows); C, collagen. The right panel depicts a negative control experiment with mouse parotid gland tissue. Color images available online at www.liebertonline.com/tea

FIG. 4.

Light microscopic views of scaffolds with human PDL cells of sham-injected mice (left panels) and those treated with PTH(1–34) for 28 days (right panels). The insets demonstrate a detail at a higher magnification. In panels (A) and (B), H&E staining exhibits a more condensed connective tissue with several scaffold-lining cells (open arrows) and early signs of osteogenesis (black arrows) in PTH(1–34)-treated specimens (B). Immunohistochemical detection (DAB) of ALP (C, D), osteopontin (E, F), and osteocalcin (G, H) in the explants after 28 days. Alizarin red staining indicates areas of mineralization in sham-injected (I) and PTH-treated (J) mice; C, collagen. Color images available online at www.liebertonline.com/tea

FIG. 5.

The effect of intermittent PTH(1–34) administration on protein expression of ALP (A), osteopontin (B), and osteocalcin (C) as well as on biomineralization (D) by human PDL cells was evaluated semiquantitatively on immunostained sections as shown in Figure 4. The bar graph shows the mean±SEM for 36 specimens per experimental group. *p<0.05, **p<0.01, and ***p<0.001, PTH(1–34) group versus vehicle control.