Medicarpin, a Natural Pterocarpan, Heals Cortical Bone Defect by Activation of Notch and Wnt Canonical Signaling Pathways.

Divya Singh,Priyanka Shukla,Aijaz A. John,Kavita Singh,Dipak Purohit,Manisha Dixit,Ashutosh Raghuvanshi,Pallavi Awasthi,Mohd Nizam Mansoori,Yin Tintut,Jyoti Kureel,Chandra Prakash Gupta,Atul Goel

doi:10.1371/journal.pone.0144541

Abstract

We evaluated the bone regeneration and healing effect of Medicarpin (med) in cortical bone defect model that heals by intramembranous ossification. For the study, female Sprague–Dawley rats were ovariectomized and rendered osteopenic. A drill hole injury was generated in mid femoral bones of all the animals. Med treatment was commenced the day after and continued for 15 days. PTH was taken as a reference standard. Fifteen days post-treatment, animals were sacrificed. Bones were collected for histomorphometry studies at the injury site by micro-computed tomography (μCT) and confocal microscopy. RNA and protein was harvested from newly generated bone. For immunohistochemistry, 5μm sections of decalcified femur bone adjoining the drill hole site were cut. By μCT analysis and calcein labeling of newly generated bone it was found that med promotes bone healing and new bone formation at the injury site and was comparable to PTH in many aspects. Med treatment led to increase in the Runx-2 and osteocalcin signals indicating expansion of osteoprogenitors at the injury site as evaluated by qPCR and immunohistochemical localization. It was observed that med promoted bone regeneration by activating canonical Wnt and notch signaling pathway. This was evident by increased transcript and protein levels of Wnt and notch signaling components in the defect region. Finally, we confirmed that med treatment leads to elevated bone healing in pre-osteoblasts by co localization of beta catenin with osteoblast marker alkaline phosphatase. In conclusion, med treatment promotes new bone regeneration and healing at the injury site by activating Wnt/canonical and notch signaling pathways. This study also forms a strong case for evaluation of med in delayed union and non-union fracture cases.

Highlights

Bone possesses an inbuilt capacity of bone regeneration which is either in response to an injury or as part of skeletal development and bone remodelling[1]
Our study reveals that med treatment repairs cortical bone defect and enhances bone regeneration in Ovx osteopenic rodents by activating notch and Wnt canonical signaling pathways
An injury is created in cortical bone at femur mid-diaphysis

Summary

Introduction

Bone possesses an inbuilt capacity of bone regeneration which is either in response to an injury or as part of skeletal development and bone remodelling[1]. Bone regeneration process involves continuous remodelling throughout adult life[2]. Certain situations such as in fracture and trauma and conditions like osteoporosis, bone regeneration is required in large quantity. The most common form of bone regeneration is fracture healing [1, 2]. Indirect bone healing is the most common form where bone healing occurs by both endochondral and intramembranous ossification. In most clinical cases of bone fracture, both cortex and marrow are disrupted. Bone regeneration in these cases involves endochondral ossification and cortical bone regeneration occurs secondarily. Cortical bone healing is one such model where cortical gap bridging occurs rapidly by intramembranous ossification [4]

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Results

Conclusion