Background
Bone formation to bridge the fracture gap following skeletal fracture slows with age in both humans [1-6] and rats [7-9]. While young, 6-week-old rats reach radiographic union by 4 weeks after femoral fracture, adult, 26-week-old rats require 10 weeks, and older, 52-week-old rats need in excess of 6 months [7]. Despite this increased time to radiographic union with age, there was no increase in the time of expression of Indian hedgehog or any of the bone morphogenetic proteins in the fracture callus for adult rats [10] or for older rats [11,12]. Radiographic union for adult and older rats occurred well after the time of expression of these skeletally active cytokines [10,11]. Except for markers of osteoblast activity and bone matrix formation, few genes remain up-regulated during the time period when bone forms to bridge the fracture gap [10-12].
These earlier studies done with RT-PCR revealed a paucity of data for genes differentially expressed by age. We had hypothesized that bone formation to bridge the fracture gap would be under a negative-feedback control system. Thus, the genes which stimulate bone formation should be up-regulated in adult or older rats to attempt to accelerate their slower progression of bony healing. This was not observed in adult [10] or older [11,12] rats. Either bone formation to bridge the fracture gap is not subject to negative-feedback control, or the genes up-regulated to control this bone formation are not those normally thought of as being involved in skeletal homeostasis. This suggested the need for a wider search for genes active during the fracture reparative process.
In this project, mRNA gene expression was measured by DNA microarray technology at various time points after fracture for young, adult, and older rats. The goal was to identify genes whose expression following fracture was altered by age. Such genes may either show reduced expression, if the age-related slowing of healing is caused by inadequate expression levels, or they may show enhanced expression, in an attempt to stimulate some poorly responding pathway. Among the genes which were differentially expressed at the fracture site with age were genes related to nerve cell activity.
In this study, we explored whether abnormal mRNA expression of genes related to nerve cell activity was associated with the slowing of skeletal repair in older rats. Abnormalities in the innervation of the fracture site will slow skeletal healing clinically [13-15] and experimentally [16-18].