Department of Anatomy and Cell Biology, Temple Medical School, Philadelphia, Pennsylvania 19140, USA.
J Cell Physiol. 2010 Oct;225(1):152-67. doi: 10.1002/jcp.22208.
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 TNFalpha 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-TNFalpha treatment improved grip strength in both groups, but did not ameliorate forepaw hypersensitivity. Moreover, anti-TNFalpha treatment attenuated task-induced increases in inflammatory cytokines (TNFalpha, IL-1alpha, and MIP2 in serum; TNFalpha 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-TNFalpha treatment. CTGF levels were unaffected by task performance or anti-TNFalpha treatment in 6-week HRHF muscles. In primary osteoblast cultures, TNFalpha, 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-E1 osteoblast-like cell cultures. Thus, tissue loading in vivo resulted in increased TNFalpha, which increased PLF, which then induced anabolic bone formation, the latter results confirmed in vitro.
我们之前开发了一种自愿的大鼠高频重复伸展模型，该模型提供了一个机会，可以在体内研究非承重肌肉负荷对上肢组织的骨和天然炎症机制的影响。在这项研究中，我们使用该模型研究了炎症细胞因子与基质细胞蛋白（骨膜蛋白样因子，PLF 和结缔组织生长因子，CTGF）之间的关系。我们还检查了炎症细胞因子、PLF 和骨形成过程之间的关系。大鼠首先接受 5 周的初始训练，然后进行高频高力（HRHF）任务（12 次/分钟，60%最大握力，2 小时/天，每周 3 天）6 周。然后，我们检查了训练或任务表现的效果，以及是否使用大鼠特异性 TNFalpha 抗体治疗，对炎症细胞因子、骨钙素（骨形成标志物）、PLF、CTGF 和疼痛或不适的行为指标的影响。HRHF 任务降低了握力，并在训练对照组和 6 周 HRHF 动物中引起前爪机械性超敏反应。两周的抗 TNFalpha 治疗改善了两组的握力，但未能改善前爪超敏反应。此外，抗 TNFalpha 治疗减弱了任务诱导的炎症细胞因子（血清中的 TNFalpha、IL-1alpha 和 MIP2；前肢骨骼和肌肉中的 TNFalpha）和血清骨钙素的增加在 6 周 HRHF 动物中。6 周 HRHF 动物的前肢骨骼和屈肌肌肉中的 PLF 水平显著增加，抗 TNFalpha 治疗可减弱这种增加。在 6 周 HRHF 肌肉中，CTGF 水平不受任务表现或抗 TNFalpha 治疗的影响。在原代成骨细胞培养物中，TNFalpha、MIP2 和 MIP3a 处理以剂量依赖性方式增加 PLF 水平。同样在原代成骨细胞培养物中，增加的 PLF 促进增殖和分化，后者通过测量 Runx2、碱性磷酸酶（ALP）和骨钙素 mRNA 水平；ALP 活性；以及钙沉积和矿化来评估。在 MC3T3-E1 成骨样细胞培养物中，增加的 PLF 还促进了细胞黏附。因此，体内组织负荷导致 TNFalpha 增加，TNFalpha 增加了 PLF，随后诱导了合成代谢性骨形成，这一结果在体外得到了证实。
