Role of TNF Alpha and PLF in Bone Remodeling in a Rat Model of Repetitive Reaching and Grasping

We have previously developed a voluntary rat model of highly repetitive reaching that provides an opportunity to study effects of non-weight bearing muscular loads on bone and mechanisms of naturally occurring inflammation on upper limb tissues in vivo. In this study, we investigated the relationship between inflammatory cytokines and matricellular proteins (Periostin-like-factor, PLF, and connective tissue growth factor, CTGF) using our model. We also examined the relationship between inflammatory cytokines, PLF and bone formation processes. Rats underwent initial training for 5 weeks, and then performed a high repetition high force (HRHF) task (12 reaches/min, 60% maximum grip force, 2 h/day, 3 days/week) for 6 weeks. We then examined the effect of training or task performance with or without treatment with a rat specific TNF alpha antibody on inflammatory cytokines, osteocalcin (a bone formation marker), PLF, CTGF, and behavioral indicators of pain or discomfort. The HRHF task decreased grip strength and induced forepaw mechanical hypersensitivity in both trained control and 6-week HRHF animals. Two weeks of anti-TNF alpha treatment improved grip strength in both groups, but did not ameliorate forepaw hypersensitivity. Moreover, anti-TNF alpha treatment attenuated task-induced increases in inflammatory cytokines (TNF alpha, IL-I alpha, and MIP2 in serum; TNF alpha in forelimb bone and muscles) and serum osteocalcin in 6-week HRHF animals. PLF levels in forelimb bones and flexor digitorum muscles increased significantly in 6-week HRHF animals, increases attenuated by anti-TNF alpha treatment. CTGF levels were unaffected by task performance or anti-TNR alpha treatment in 6-week HRHF muscles. In primary osteoblast cultures, INF alpha, MIP2 and MIP3a treatment increased PLF levels in a dose dependent manner. Also in primary osteoblast cultures, increased PLF promoted proliferation and differentiation, the latter assessed by measuring Runx2, alkaline phosphatase (ALP) and osteocalcin mRNA levels; ALP activity; as well as calcium deposition and mineralization. Increased PLF also promoted cell adhesion in MC3T3-EI osteoblast-like cell cultures. Thus, tissue loading in vivo resulted in increased TNF alpha, which increased PLF, which then induced anabolic bone formation, the latter results confirmed in vitro. J. Cell. Physiol. 225: 152-167, 2010. (C) 2010 Wiley-Liss, Inc.