Effect of parathyroid hormone-related protein in an in vitro hypertrophy model for mesenchymal stem cell chondrogenesis

Abstract

Purpose: Mesenchymal stem cells (MSCs) express markers of hypertrophic chondrocytes during chondrogenic differentiation. We tested the suitability of parathyroid hormone-related protein (PTHrP), a regulator of chondrocyte hypertrophy in embryonic cartilage development, for the suppression of hypertrophy in an in vitro hypertrophy model of chondrifying MSCs.

Methods: Chondrogenesis was induced in human MSCs in pellet culture for two weeks and for an additional two weeks cultures were either maintained in standard chondrogenic medium or transferred to a hypertrophy-enhancing medium. PTHrP(1-40) was added to the medium throughout the culture period at concentrations from 1 to 1,000 pM. Pellets were harvested on days one, 14 and 28 for biochemical and histological analysis.

Results: Hypertrophic medium clearly enhanced the hypertrophic phenotype, with increased cell size, and strong alkaline phosphatase (ALP) and type X collagen staining. In chondrogenic medium, 1-100 pM PTHrP(1-40) did not inhibit chondrogenic differentiation, whereas 1,000 pM PTHrP(1-40) significantly reduced chondrogenesis. ALP activity was dose-dependently reduced by PTHrP(1-40) at 10-1,000 pM in chondrogenic conditions. Under hypertrophy-enhancing conditions, PTHrP(1-40) did not inhibit the induction of the hypertrophy. At the highest concentration (1,000 pM) in the hypertrophic group, aggregates were partially dedifferentiated and differentiated areas of these aggregates maintained their hypertrophic appearance.

Conclusions: PTHrP(1-40) treatment dose-dependently reduced ALP expression in MSC pellets cultured under standard chondrogenic conditions and is thus beneficial for the maintenance of the chondrogenic phenotype in this medium condition. When cultured under hypertrophy-enhancing conditions, PTHrP(1-40) could not diminish the induced enhancement of hypertrophy in the MSC pellets.

Fig. 1

Biochemical analysis. Effect of PTHrP(1–40) treatment on DNA (a) and GAG (b) content and ALP secretion (c) in standard chondrogenic medium (CM) and hypertrophy-enhancing medium (Hyp) on day 28. GAG contents are expressed normalised to DNA contents. Values shown are mean ± SD (n = 4); *significantly different from control without PTHrP(1–40); +significant difference between chondrogenic and hypertrophic group at the respective PTHrP(1–40) concentration (p < 0.05); a and b: n = 4; c: n = 8

Fig. 2

DMMB staining on day 28 of chondrogenic (a–e) and hypertrophic (f–j) aggregates without (a, f) and with 1 pM (b, g), 10 pM (c, h), 100 pM (d, i) and 1,000 pM PTHrP(1–40) (e, j). jBlack arrows dedifferentiated peripheral ring without metachromasia, white arrow low DMMB staining in the bottom third of the aggregate. Magnification ×100, bar 100 μm

Fig. 3

Type II collagen immunohistochemistry. Immunohistochemical staining for type II collagen on day 28 of chondrogenic (a–e) and hypertrophic (f–j) aggregates without (a, f) and with 1 pM (b, g), 10 pM (c, h), 100 pM (d, i) and 1,000 pM PTHrP(1–40) (e, j). Arrows indicate dedifferentiated type II collagen-negative areas in f. Magnification ×100, bar 100 μm

Fig. 4

Type X collagen immunohistochemistry. Immunohistochemical staining for type X collagen on day 28 of chondrogenic (a–e) and hypertrophic (f–j) aggregates without (a, f) and with 1 pM (b, g), 10 pM (c, h), 100 pM (d, i) and 1,000 pM PTHrP(1–40) (e, j). Arrows indicate dedifferentiated type X collagen-negative areas in f. Magnification ×100, bar 100 μm

Fig. 5

ALP histochemistry. Histochemical ALP staining on day 28 of chondrogenic (a–e) and hypertrophic (f–j) aggregates without (a, f) and with 1 pM (b, g), 10 pM (c, h), 100 pM (d, i) and 1,000 pM PTHrP(1–40) (e, j). Blue ALP-positive areas, red neutral red counterstaining. Arrows in f indicate areas that represent dedifferentiated regions without the typical cartilage-like morphology. Magnification ×100, bar 100 μm

Fig. 6

Possible effects of PTHrP in the model used. PTHrP inhibits chondrogenic differentiation of MSCs at high concentrations (1). After chondrogenic conditioning when hypertrophy is induced by switching the medium conditions, PTHrP seems to promote dedifferentiation (2) and may inhibit terminal differentiation towards a hypertrophic phenotype (3). ALP activity in the periphery of the aggregates is possibly induced by chondrocytes and hypertrophic chondrocytes in the centre of the aggregates and PTHrP may indirectly inhibit ALP induction in the periphery by inhibiting chondrogenesis and maturation (4). PTHrP may directly suppress ALP expression in the peripheral non-chondrogenic cells (5)