eCM (Eur Cell Mater / e Cells & Materials) Not-for-Profit Open Access
Created by Scientists, for Scientists
 ISSN:1473-2262         NLM:100973416 (link)         DOI:10.22203/eCM

2020   Volume No 39 – pages 183-192

Title: Enhancement of fracture healing after citrulline supplementation in mice

Authors: DM Meesters, PFW Hannemann, HMH van Eijk, VTJ Schriebl, PRG Brink, M Poeze, KAP Wijnands

Address: Maastricht University Medical Centre, Department of Surgery and Trauma Surgery, PO Box 5800, 6202 AZ Maastricht, the Netherlands

E-mail: d.meesters at

Abstract: Around 10 % of long bone fractures show inadequate bone healing resulting in non-union development. A deregulated arginine-citrulline-nitric oxide metabolism caused by a poor nutritional status of the patients is a risk factor for non-unions. Additionally, previous research in mice with a disrupted arginine to citrulline conversion showed delayed healing. The study hypothesis was that stimulating said metabolism could positively influence the healing process through promotion of collagen synthesis and angiogenesis.
     Adult wild-type mice underwent a femur osteotomy and plate-screw osteosynthesis. Mice were randomly divided into three groups and received daily oral supplementation of arginine, citrulline or 0.9 % saline (control). Body weight and food intake were measured daily. After 14 d, the mice were euthanised and femora collected. Callus formation was assessed by micro-computed tomography and concentrations of amino acids and enzymes in the femora were measured.
     Only citrulline-treated mice showed significantly increased bridging of the fracture gap when compared to control mice. Femur citrulline and ornithine concentrations were increased in citrulline-treated animals. qPCR showed significantly decreased expression of inflammatory markers, whereas increased expression of angiogenic and collagen-producing factors was observed in citrulline-treated mice. Although food intake did not show any difference between the three groups, animals treated with citrulline showed a weight gain of 0.3 g, compared with a 0.1 g decline in the control group.
     Daily oral citrulline supplementation stimulated callus formation and improved the inflammatory response, positively contributing to the enhanced healing response. Finally, the increased weight gain pointed toward a better post-operative recovery.

Key Words: Non-union, citrulline, arginine, nitric oxide, fracture healing.

Publication date: March 20th 2020

Article download: Pages 183-192 (PDF file)

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