Low frequency EMF regulates chondrocyte differentiation and expression of matrix proteins

Abstract

This study describes the enhancement of chondrogenic differentiation in endochondral ossification by extremely low frequency pulsed electric/magnetic fields (EMFs). The demineralized bone matrix (DBM)-induced endochondral ossification model was used to examine the effects of EMF stimulation. [ 35 S ] -Sulfate and [ 3 H ] -thymidine incorporation and glycosaminoglycan (GAG) content were determined by standard methods. Proteoglycan (PG) and GAG molecular size and composition were determined by gel chromatography and sequential enzyme digestion. Immunohistochemical and Western blot analysis of PGs were done with antibodies 2B6, 3B3, 2D3 and 5D4. Northern analysis of total RNA extracts was performed for aggrecan, and type II collagen. All data was compared for significance by Student's t - or analysis of variance (ANOVA)-tests. The EMF field accelerated chondrogenesis as evidenced by an increase in: (1) 35 SO 4 incorporation and GAG content, (2) the number of chondrocytes at day 8 of development, (3) the volumetric density of cartilage and (4) the extent of immunostaining for 3B3 and 5D4. No differences in DNA content or [ 3 H ] -thymidine incorporation were observed between control and stimulated ossicles, suggesting the absence of enhanced cell proliferation or recruitment as a mechanism for the acceleration. PG and GAG molecular sizes and GAG chemical composition were similar in stimulated and control ossicles, indicating that stimulation resulted in an accelerated synthesis of normal cartilage molecules. The increased expression of PG and type II collagen mRNA as well as a greater immunoreactivity of 3B3 and 5D4 suggest an increase in the rate of differentiation of chondrocytes and enhanced phenotypic maturation.