Deficiencies of folate and vitamin B12 do not affect fracture healing in mice

Abstract

Purpose

Recently, hyperhomocysteinemia has been shown to be associated with impaired fracture healing in mice. The main causes for hyperhomocysteinemia are deficiencies of folate and vitamin B12. However, there is no information on whether deficiencies of these B vitamins are affecting bone repair, too.

Methods

We used two groups of mice to investigate the impact of folate and vitamin B12 deficiency on fracture healing: mice of the first group were fed a folate- and vitamin B12-deficient diet (n = 14), while mice of the second group received an equicaloric control diet (n = 13). Four weeks after stabilizing a closed femur fracture, bone repair was analyzed by histomorphometry and biomechanical testing. In addition, serum concentrations of homocysteine, folate, vitamin B12, the bone formation marker osteocalcin (OC), and the bone resorption marker collagen I C-terminal crosslaps (CTX) were measured.

Results

Serum analyses revealed significantly decreased concentrations of folate and vitamin B12 in animals fed the folate- and vitamin B12-deficient diet when compared to controls. This was associated with a moderate hyperhomocysteinemia in folate- and vitamin B12-deficient mice, while no hyperhomocysteinemia was found in controls. Three-point bending tests showed no significant differences in callus stiffness between bones of folate- and vitamin B12-deficient animals and those of control animals. In accordance, the histomorphometric analysis demonstrated a comparable size and tissue composition of the callus, and also serum markers of bone turnover did not differ significantly between the two groups.

Conclusions

We conclude that folate and vitamin B12 deficiency does not affect bone repair in mice.

Introduction

In the Western civilization hyperhomocysteinemia (HHCY) represents a widespread metabolic disorder leading to cardiovascular disease and osteoporosis [1], [2]. Homocysteine (HCY) is a metabolite of the essential amino acid methionine [3]. Folate, vitamin B6, and vitamin B12 are essential co-enzymes of the HCY degrading remethylation and trans sulfuratin pathways [3]. In accordance, deficiencies of B vitamins are among the main causes of elevated HCY serum concentrations [4], [5]. In countries without folate supplementation of food, the prevalence of B vitamin deficiencies ranges between 20% and 45% [6], [7], [8].

Several clinical studies as well as some recent animal studies have indicated HHCY and B vitamin deficiency to be associated with an impaired bone quality [9], [10], [11], [12], [13], [14], [15]. Of interest, an additional animal study in mice has recently demonstrated that HHCY adversely affects also fracture healing [16]. In this study, HHCY was induced by a HCY-supplemented diet. Using a closed femur fracture model bone repair was analyzed at 4 weeks. Biomechanical testing of the healing bones revealed a significantly lower callus stiffness, while histomorphometric analyses showed a slightly smaller callus diameter in hyperhomocysteinemic animals when compared to normohomocysteinemic controls.

The induction of HHCY by a HCY-supplemented diet represents a relatively artificial model of HHCY, as B vitamin deficiency rather than an alimentary HCY overload is the most common cause of HHCY in humans. In accordance, the herein introduced study was conducted to investigate on whether also a folate- and vitamin B12-deficient diet might affect bone repair in mice.