Deficiencies of folate and vitamin B6 and B12 as well as increased methionine serum concentrations have been indicated to disturb bone metabolism, most probably due to an induction of hyperhomocysteinemia (HHCY). However, there is a complete lack of information on whether these metabolic changes affect fracture healing. Therefore, the aim of this study was to analyze the impact of a methionine-enriched (n=13) and a B vitamin-deficient diet (n=14) on bone repair in mice. Controls were fed by the accordant standard diet (n=12 and n=13). Four weeks after stable fixation of a closed femoral fracture, animals were sacrificed to prepare bones for histomorphometric and biomechanical analyses. In addition, blood samples were obtained to evaluate serum concentrations of homocysteine (HCY), folate, and vitamin B12. Quantitative analysis of blood samples revealed significantly increased serum concentrations of HCY associated with significantly decreased serum concentrations of folate and vitamin B12 in animals fed with the methionine-enriched diet or the B vitamin-deficient diet when compared to controls. Biomechanical evaluation showed no significant differences in bending stiffness between bones of the experimental and those of the control groups. In accordance, the histomorphometric analysis demonstrated a comparable size and tissue composition of the callus in all groups analyzed. We conclude that a methionine-enriched and a B vitamin-deficient diet leads to HHCY, however, without affecting bone repair in mice.
A major pathway of closed soft-tissue injury is failure of microvascular perfusion combined with a persistently enhanced inflammatory response. We therefore tested the hypothesis that hypertonic hydroxyethyl starch (HS/HES) effectively restores microcirculation and reduces leukocyte adherence after closed soft-tissue injury. We induced closed soft-tissue injury in the hindlimbs of 14 male isoflurane-anaesthetised rats. Seven traumatised animals received 7.5% sodium chloride-6% HS/HES and seven isovolaemic 0.9% saline (NS). Six non-injured animals did not receive any additional fluid and acted as a control group. The microcirculation of the extensor digitorum longus muscle (EDL) was quantitatively analysed two hours after trauma using intravital microscopy and laser Doppler flowmetry, i.e. erythrocyte flux. Oedema was assessed by the wet-to-dry-weight ratio of the EDL. In NS-treated animals closed soft-tissue injury resulted in massive reduction of functional capillary density (FCD) and a marked increase in microvascular permeability and leukocyte-endothelial cell interaction as compared with the control group. By contrast, HS/HES was effective in restoring the FCD to 94% of values found in the control group. In addition, leukocyte rolling decreased almost to control levels and leukocyte adherence was found to be reduced by ~50%. Erythrocyte flux in NS-treated animals decreased to 90 ± 8% (mean HS/HES effectively restores nutritive perfusion, decreases leukocyte adherence, improves endothelial integrity and attenuates oedema, thereby restricting tissue damage evolving secondary to closed soft-tissue injury. It appears to be an effective intervention, supporting nutritional blood flow by reducing trauma-induced microvascular dysfunction.
