Development of a cyclic-inverso AHSG/Fetuin A-based peptide for inhibition of calcification in osteoarthritis.

OBJECTIVE: Ectopic calcification is an important contributor to chronic diseases, such as osteoarthritis. Currently, no effective therapies exist to counteract calcification. We developed peptides derived from the calcium binding domain of human Alpha-2-HS-Glycoprotein (AHSG/Fetuin A) to counteract calcification. METHODS: A library of seven 30 amino acid (AA) long peptides, spanning the 118 AA Cystatin 1 domain of AHSG, were synthesized and evaluated in an in vitro calcium phosphate precipitation assay. The best performing peptide was modified (cyclic, retro-inverso and combinations thereof) and evaluated in cellular calcification models and the rat Medial Collateral Ligament Transection + Medial Meniscal Tear (MCLT + MMT) osteoarthritis model. RESULTS: A cyclic peptide spanning AA 1-30 of mature AHSG showed clear inhibition of calcium phosphate precipitation in the nM-pM range that far exceeded the biological activity of the linear peptide variant or bovine Fetuin. Biochemical and electron microscopy analyses of calcium phosphate particles revealed a similar, but distinct, mode of action in comparison with bFetuin. A cyclic-inverso variant of the AHSG 1-30 peptide inhibited calcification of human articular chondrocytes, vascular smooth muscle cells and during osteogenic differentiation of bone marrow derived stromal cells. Lastly, we evaluated the effect of intra-articular injection of the cyclic-inverso AHSG 1-30 peptide in a rat osteoarthritis model. A significant improvement was found in histopathological osteoarthritis score and animal mobility. Serum levels of IFNγ were found to be lower in AHSG 1-30 peptide treated animals. CONCLUSIONS: The cyclic-inverso AHSG 1-30 peptide directly inhibits the calcification process and holds the potential for future application in osteoarthritis.

Enhancement of fracture healing after citrulline supplementation in mice.

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.

Deficiency of inducible and endothelial nitric oxide synthase results in diminished bone formation and delayed union and nonunion development.

BACKGROUND: Between 5% and 10% of all fractures fail to heal adequately resulting in nonunion of the fracture fragments. This can significantly decrease a patient's quality of life and create associated psychosocial and socio-economic problems. Nitric oxide (NO) and nitric oxide synthases (NOS) have been found to be involved in fracture healing, but until now it is not known if disturbances in these mechanisms play a role in nonunion and delayed union development. In this study, we explored the role of endothelial and inducible NOS deficiency in a delayed union model in mice. MATERIALS AND METHODS: A 0.45mm femur osteotomy with periosteal cauterization followed by plate-screw osteosynthesis was performed in the left leg of 20-24week old wild type, Nos2(-/-) and Nos3(-/-) mice. Contralateral unfractured legs were used as a control. Callus volume was measured using micro-computed tomography (µCT) after 28 and 42days of fracture healing. Immuno histochemical myeloperoxidase (MPO) staining was performed on paraffin embedded sections to assess neutrophil influx in callus tissue and surrounding proximal and distal marrow cavities of the femur. After 7 and 28days of fracture healing, femurs were collected for amino acid and RNA analysis to study arginine-NO metabolism. RESULTS: With µCT, delayed union was observed in wild type animals, whereas in both Nos2(-/-) and Nos3(-/-) mice nonunion development was evident. Both knock-out strains also showed a significantly increased influx of MPO when compared with wild type mice. Concentrations of amino acids and expression of enzymes related to the arginine-NO metabolism were aberrant in NOS deficient mice when compared to contralateral control femurs and wild type samples. DISCUSSION AND CONCLUSION: In the present study we show for the first time that the absence of nitric oxide synthases results in a disturbed arginine-NO metabolism and inadequate fracture healing with the transition of delayed union into a nonunion in mice after a femur osteotomy. Based on these data we suggest that the arginine-NO metabolism may play a role in the prevention of delayed unions and nonunions.