Low dose erythropoietin stimulates bone healing in mice

Abstract

Beyond its classical role in regulation of erythropoiesis, erythropoietin (EPO) has been shown to exert protective and regenerative actions in a variety of non-hematopoietic tissues. However, little is known about potential actions in bone regeneration. To analyze fracture healing in mice, a femoral 0.25 mm osteotomy gap was stabilized with a pin-clip technique. Animals were treated with 500 U EPO/kg bw per day or with vehicle only. After 2 and 5 weeks, fracture healing was analyzed biomechanically, radiologically and histologically. Expression of PCNA and NFB was examined by Western blot analysis. Vascularization was analyzed by immunohistochemical staining of PECAM-1. Circulating endothelial progenitor cells were measured by flow-cytometry. Herein, we demonstrate that EPOtreatment significantly accelerates bone healing in mice. This is indicated by a significantly greater biomechanical stiffness and a higher radiological density of the periosteal callus at 2 and 5 weeks after fracture and stabilization. Histological analysis demonstrated significantly more bone and less cartilage and fibrous tissue in the periosteal callus. Endosteal vascularization was significantly increased in EPO-treated animals when compared to controls. The number of circulating endothelial progenitor cells was significantly greater in EPO-treated animals. The herein shown acceleration of healing by EPO may represent a promising novel treatment strategy for fractures with delayed healing and non-union formation. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J. Orthop. Res. 29: 165-172, 2011 Keywords: mouse; erythropoietin; fracture healing; bone healing; bone regeneration Beyond its role in the regulation of red blood cell proliferation, the glycoprotein erythropoietin (EPO) has been shown to exert protective and regenerative actions in a variety of non-hematopoietic tissues. In a previous study we could show that daily application of 5,000 U/kg EPO for 5 days (high-dose, short-time) enhances early endochondral ossification and mechanical strength in a closed femoral fracture model in mice. 2 However, the initial effect of short-time and high-dose application of EPO was found vanished after 5 weeks, most probably because of the good healing response of closed fractures in mice and the limitation of EPO administration to only 5 days post fracture. In the present study we analyzed, whether EPO is capable of accelerating healing of fractures with small segmental defects in mice. Because several studies have shown that high EPO doses of up to 5,000 U/kg/day are not necessary to achieve tissue protection and that this high doses can be associated with adverse effects, 3 we have studied low-dose treatment with only 500 U/kg/day EPO. Further, because the EPO-receptor has been shown to be expressed within the periosteal callus over several weeks during fracture healing, 2 EPO was given in the present study as a long term treatment over a period of 5 weeks. MATERIALS AND METHODS AnimalsA total of 52 CD-1 m...

The effect of erythropoietin on bone

“…In agreement with the findings of this thesis, the majority of the published pre-clinical studies document an advantageous effect of EPO on early bone healing (Holstein et al 2007;Garcia et al 2011;. Taken together with the first data from a clinical trial, these findings indicate an improved healing process and (Bakhshi et al 2013).…”

Erythropoietin Modulates the Structure of Bone Morphogenetic Protein 2–Engineered Cranial Bone

“…It was reported that Epo at a dose of 500 U/kg significantly increased the fracture healing in a mouse femoral osteotomy model. 47 However, 300 U/kg and 10-day intraperitoneal injection of Epo was found to decrease the trabecular BV in mouse tibia. 46 Therefore, the dosage, duration, and route for Epo administration, and the anatomic site of bone to be regenerated may also be important considerations for the application of Epo for bone tissue engineering.…”

“…23 Recently, one group reported Epo to improve long bone healing, possibly due to its angiogenic effects. 47,58,59 Despite these findings, the role of Epo in bone repair remains largely unknown.…”

Inhibition of adipogenic differentiation of bone marrow mesenchymal stem cells by erythropoietin via activating ERK and P38 MAPK

“…The role of EPO in non-hematopoietic cells includes promotion of endothelial cell proliferation (Anagnostou et al, 1994), protection of myocardial injury caused by myocardial infarction (Kagaya et al, 2012), inhibition of ventricular remodeling induced by angiotensin II (Zhang et al, 2009), and improvement of the recovery of damaged nerve cells (Sirén et al, 2001). In addition, EPO can inhibit inflammatory factors and promote fracture healing (Garcia et al, 2011) by reducing the expression of nuclear factor-kB. Hojman et al (2009) demonstrated that EPO can inhibit the weight gain of mice induced by a high-fat diet, while mice lacking EPO-R expression are more likely to gain fat and develop insulin resistance.…”