Differences in Osteoclastogenic Regulatory Factors in Pediatric and Adult Subjects

Objectives: Differentiation of osteoclasts is regulated by factors produced by osteoblasts and marrow stromal cells. To test the hypothesis that the regulators would be higher in pediatric than adult groups, we compared  expression of the pro-osteoclastogenic factors Receptor Activator of Nuclear Factor-kB-Ligand (RANKL) and M-CSF and the anti-osteoclastogenic factor osteoprotegerin (OPG) in marrow-derived stromal cells.

Methods: With IRB approval and annual review, pediatric bone marrow was obtained from excess iliac crest bone graft harvested for cleft palate repair, and adult bone marrow was obtained as tissue discarded during total hip replacement for non-inflammatory osteoarthritis. Low-density marrow cells from 8 pediatric (ages 8-17, mean 11) and 10 adult (ages 43-50, mean 47) subjects were isolated by centrifugation with Ficoll/Histopaque 1077. Aliquots were cultured in phenol red-free α-MEM medium. Non-adherent hematopoietic cells were removed after 24 hours, and adherent marrow stromal cells were expanded to passage 2. Upon near confluence, total RNA was isolated with Trizol reagent. Expression of RANKL, M-CSF, OPG, and housekeeping gene GAPDH was measured by reverse transcription-PCR. Products were separated by electrophoresis, quantified by densitometry, and expressed relative to GAPDH. Group data are presented as mean ± SEM, and were analyzed with Mann-Whitney test or unpaired t-test, when appropriate.

Results: There were significant differences in expression of osteoclast regulatory factors between the age groups. Expression of RANKL was 2.5-fold greater in pediatric than adult subjects (p=0.021). Expression of OPG was 2.6-fold greater in pediatric than adult subjects (p<0.0001). No significant difference in expression of M-CSF was found.

Gene	Pediatric (8)	Adult (10)	P value
RANKL/GAPDH	1.03±0.12	0.42±0.20	0.021(Mann-Whitney)
OPG/GAPDH	0.66±0.05	0.25±0.04	<0.0001(t-test)
M-CSF/GAPDH	1.22±0.08	1.01±0.15	NS (Mann-Whitney)

 

 

Conclusions: Increased expression of RANKL and OPG in stromal cells from pediatric subjects is consistent with greater bone modeling in growing children. This study supports age-dependent differences in expression of osteoclastogenic regulatory factors.