Peripheral nerve extract effects on mesenchymal cells

Several common congenital limb disorders are characterized by normal tissue differentiation but abnormal somatic growth. These include: idiopathic clubfoot, idiopathic leg length discrepancy, hemi-atrophy and hemi-hypertrophy. Both clinical and research studies have suggested that peripheral nerves may be important in regulating somatic growth of limb tissues. To investigate the hypothesis that peripheral nerves convey trophic substances to mesenchymal tissues that are involved in the regulation of growth, we developed an in vitro assay to assess the effect of fractions of peripheral nerve on myoblast and chondroblast growth and differentiation in a mammalian (rat) system. Whole rat sciatic nerve extract was fractionated by ammonium sulfate precipitation and by affinity chromatography. Concavalin A chromatography resolved whole nerve extract into a glycoprotein and a non-glycoprotein fraction. Serial ammonium sulfate precipitation yielded three pellet fractions designated as 35%, 70%, and 100% pellets; corresponding to ammonium sulfate concentrations of 0 to 35%, 35 to 70%, and 70 to 100% saturation, respectively. Dialyzed solutions of these pellets as well as the fractions from Concavalin A chromatography were assayed for biological activity in micromass cultures of rat limb bud mesenchyme, which allowed assessment of both myoblast and chondroblast stimulation. Stimulation of protein synthesis and myoblast proliferation (as measured by MF20 staining) occurred with both 70% and 100% ammonium sulfate fractions. Stimulation of chondroblasts (as measured by the number of alcian blue staining nodules) occurred with the 35% and 100% fractions. The glycoprotein fraction from the affinity chromatography stimulated protein synthesis and myoblast proliferation and inhibited chondroblast development. Stimulation of chondroblasts was seen with the non-glycoprotein fraction. No effect on protein synthesis, myoblast proliferation or chondroblast proliferation was found in cultures treated with rat transferrin (transferrin has been reported to stimulate myoblasts in avian culture systems).