The adaptation of bone to mechanical loading is very poorly understood at the molecular genetic level. Little is known of gene expression in bone following mechanical stimulation primarily because of a lack of in vivo data. While the calcified extracellular matrix provides bone with its mechanical strength, it also makes the analysis of genetic information difficult. A new method has been developed which can extract high quality RNA from intact bone. This new technique enables the use of the Sprague Dawley rat to study the in vivo regulation of skeletal gene expression.

The tibia of fifteen skeletally mature female rats were subjected to either a dynamic four point bending stimulation or parathyroid hormone (PTH) stimulation. The animals were sacrificed within a twenty-four hour period following stimulation. The RNA within the tibia was extracted with the novel method and examined by Northern blot analysis. The constitutive expression of mRNA transcripts coding for transforming growth factor-beta 1 (TGF-β1) and α-1 collagen I (COL I α-1) were observed. Densitometric examinations demonstrated that over the first 24 hours, there was down regulation of TGF-β1 mRNA following mechanical stimulation (p = 0.005). The TGF-β1 mRNA was maximally depressed at 1 hour and increased gradually over the remaining twenty-four hours. PTH seemed to cause a similar down regulation of TGF-β1 mRNA but no significance was reached (p = 0.097).

To better quantify transcriptional differences, an internal control gene, ribosomal protein S3 (RPS3), was selected. RPS3 is transcribed at a steady state rate and can therefore be used to normalize other mRNA levels. With this technique, a six fold increase in COL I α-1 mRNA transcripts was observed twelve hours following stimulation with PTH (p =.006).

The constitutive expression of TGF-β1 and COL I α-1 in bone in vivo are significant new pieces of molecular genetic information. TGF-β1 and COL I α-1 are factors known to be involved in the process of bone formation. The fact that they are always present indicates that their in vivo regulated expression may prove helpful in our understanding of the mechanisms of human bone formation.