In this study we have measured mRNA gene expression in female rats for six weeks after femoral fracture. Females were chosen because female rats do not grow as large as male rats [22
]. This then minimized the difference in body size between the two ages. Young mammals rapidly progress to fracture healing as we [11
] and others [13
] have shown before. In contrast, older rats and older humans heal more slowly [11
]. In our earlier studies, comparable rats at one year of age failed to regain normal skeletal biomechanics within six months of femoral fracture, while younger rats regained normal biomechanics within four weeks after fracture [11
]. In the present study the goal was to examine possible alterations in expression of genes related to the BMPs in order to explain this failure of the older rats to heal promptly. Despite the difference in age, all genes studied increased in expression after fracture followed by a decrease to baseline values by 4 weeks after fracture.
This decrease in the mRNA gene expression to undetectable levels at 4-6 weeks after fracture was the major conclusion of this study. The decrease occurred in rats of both ages and was independent of the healing status of the fractures. This was a surprising result, and occurred for both matrix and regulatory genes. It was unexpected since the genes under study were genes needed for fracture healing. We had anticipated that the older rats would have an up-regulation of regulatory gene expression to attempt to accelerate the healing process. We speculate that this decrease in gene expression is responsible for the failure of healing to progress more quickly in the older rats.
Our results appear to violate the principle of negative feedback control over biological processes. A slower healing sequence in older individuals might be caused by a diminished number of cells available to effect the repair [16
]. This inadequate cellular response should lead to enhanced cytokine signaling by the BMPs to attempt to elicit a healing response by the bone cells. The enhanced signal should persist until healing is complete. The failure of enhanced BMP expression at four and six weeks after fracture in the older rats is difficult to explain. It may suggest that there is a constant time of stimulation of cytokine production following injury, rather than negative feedback control over the process of fracture healing. Injury may initiate a stimulus for bone healing that leads to enhanced BMP production for only a short time. For the younger rats, this time period seems adequate to allow healing to occur. For the older rats, a more prolonged signal may be required. The evaluation of this hypothesis will require additional experimentation.
There are changes in the periosteal cell layer with advancing age [16
]. Following fracture there is an increase in the rate of mitosis in the periosteum near the fracture site. This rate is altered with age: Fewer cells enter mitosis, and more time is required for the cells to undergo mitosis [16
]. This may reflect a reduction in the number of osteogenic stem cells available for skeletal repair in older individuals [17
]. Despite this, we have extracted a similar amount of mRNA from the fracture site, with a similar increase in gene expression, in both the older and the younger rats. There was no evidence for a failure of gene expression for any of the genes studied thus far in the fracture callus of older rats.
The role of the BMPs has been studied in fracture healing in rats [2
], mice [4
], and rabbits [6
] and in healing of spinal fusions in rabbits [9
]. For the most part, these studies were done in younger animals who heal their bone fractures quickly. The rise in expression of the BMPs in these animals persists for the entire length of fracture healing through the point of formation of bridging callus. By correlating the gene up-regulation to the stage of fracture healing, it gives the impression that the BMPs are regulating all stages of fracture healing. In contrast, we have used age as an experimental model to slow the process of fracture healing [11
]. This serves to separate the various radiographic stages of fracture healing and allows us to associate progression of healing with specific sets of up-regulated genes. In the present study, expression of BMP-2 and BMP-4, along with the type IA BMP receptor, decreased to baseline prior to the formation of bridging callus. This suggests that there may be a constant time of up-regulation of gene expression following fracture in the rat. Fracture is followed by four weeks of up-regulation of the stimulatory cytokines after fracture, and healing must take place within that window of time if it is to occur. The older rats cannot respond that quickly and fail to complete the healing process. Alternatively, these cytokines may only be needed for the formation of the soft callus. Once the soft callus is formed, these genes regress to baseline. Other genes, not yet identified, may be needed to stimulate the formation of bridging callus on the scaffold of cartilage and fibrous tissue formed as part of the soft callus. If the latter hypothesis is correct, there should be expression of other cytokines late in the healing process to stimulate bone formation. We are currently engaged in a broader search for such genes.
In the unfractured bone samples, gene expression was undetectable in the older rats for all of the measured genes shown in the Figures. An exception to this was osteocalcin for which there was a low basal level of gene expression in the older rats. In contrast, the unfractured bones from the young rats had clearly detectable amplimers for osteocalcin, type I collagen, and the type IA BMP receptor. This higher level of basal gene expression reflected the greater growth rate in the diaphyseal bone of the younger rats. Unfractured bone of both ages lacked detectable amplimers for type II collagen, reflecting the lack of appreciable cartilage synthesis in the diaphysis of the long bones.
There was a positive 18S rRNA signal in each sample. This is evidence that the failure to detect amplimers for both the matrix genes and the BMP-related genes at 4 and 6 weeks after fracture was related to decreased mRNA expression of these genes. The positive 18S signal would argue against technical difficulties in detecting expression in these samples. The unstable expression of housekeeping genes after fracture is not surprising. There is considerable increase in metabolic activity by the bone cells in response to fracture, and there is also a change in the cell population of the fracture callus in comparison to intact diaphyseal bone. It is not unusual to see change in the expression of housekeeping genes with large changes in cell activity [23
We have considered whether instability could be impeding fracture healing in these older rats, and we cannot find evidence of it. All rats have intramedullary fixation. In addition, there has been no evidence of a hypertrophic callus caused by instability in the femora of the older rats that fail to reach union. Instead, after the fracture is induced, there is a slowing in the formation of the soft callus as revealed by the later peak in type II collagen. There is a more profound slowing of the bony reaction on the periosteal surface of the femoral diaphysis. The young rats have a visible periosteal reaction at week 1, while the older rats do not form mineralized tissue on the periosteal surface until the fourth week after fracture. Even if instability were present, this would not impede cytokine formation since, in a mouse model, unstable tibial fractures were associated with prolonged expression of Indian hedgehog and BMP-6 [27
These findings have led us to hypothesize inadequate cytokine signaling in the older rats to explain the delayed fracture healing. The number of animals used in the present study, while adequate to test for early decreased gene expression in the older rats, was inadequate to compare each individual time point between the two ages. This is currently being tested in a larger experiment.