Elsevier

Bone

Volume 32, Issue 4, April 2003, Pages 387-396
Bone

Original article
Temporal and spatial expression of bone morphogenetic proteins in extracorporeal shock wave-promoted healing of segmental defect

https://doi.org/10.1016/S8756-3282(03)00029-2Get rights and content

Abstract

Extracorporeal shock wave (ESW) is a noninvasive acoustic wave, which has recently been demonstrated to promote bone repair. The actual healing mechanism triggered by ESW has not yet been identified. Bone morphogenetic proteins (BMP) have been implicated as playing an important role in bone development and fracture healing. In this study, we aimed to examine the involvement of BMP-2, BMP-3, BMP-4, and BMP-7 expression in ESW promotion of fracture healing. Rats with a 5-mm segmental femoral defect were given ESW treatment using 500 impulses at 0.16 mJ/mm2. Femurs and calluses were subjected to immunohistochemistry and RT-PCR assay 1, 2, 4, and 8 weeks after treatment. Histological observation demonstrated that fractured femurs received ESW treatment underwent intensive mesenchymal cell aggregation, hypertrophic chondrogenesis, and endochondral/intramembrane ossification, resulting in the healing of segmental defect. Aggregated mesenchymal cells at the defect, chondrocytes at the hypertrophic cartilage, and osteoblasts adjunct to newly formed woven bone showed intensive proliferating cell nuclear antigen expression. ESW treatment significantly promoted BMP-2, BMP-3, BMP-4, and BMP-7 mRNA expression of callus as determined by RT-PCR, and BMP immunoreactivity appeared throughout the bone regeneration period. Mesenchymal cells and immature chondrocytes showed intensive BMP-2, BMP-3, and BMP-4 immunoreactivity. BMP-7 expression was evident on osteoblasts located at endochondral ossification junction. Our findings suggest that BMP play an important role in signaling ESW-activated cell proliferation and bone regeneration of segmental defect.

Introduction

Fracture healing is a complex process involving the growth and differentiation of osteogenic progenitor cells, the regulation of inflammatory cytokines, and the synthesis and resorption of the extracellular matrix [1], [2]. Whereas, a nonunion fracture is a complicated musculoskeletal disease evidenced by a failure of new bone to bridge the defect or a development of the extracellular matrix into loose fibrous tissues or fibrocartilage [3]. Several biophysical methods, such as low-intensity ultrasound, pulsed electric magnetic field, and mechanical loading, have been identified with the promotion of bone formation and fracture healing [4], [5], [6]. Extracorporeal shock wave (ESW) is able to release pulsed acoustic energy and has been shown to be an alternative, noninvasive biophysical strategy to enhance fracture healing [7], [8], [9]. ESW treatment has previously been shown to affect local blood flow and bone metabolism in rabbit femur [10]. However, the exact mechanism by which ESW promotes fracture healing remains undetermined.

Differentiation of mesenchymal cells and endochondral ossification into the shape of future skeletal elements has been implicated as recapitulation of embryonic skeletal development in the repair of bone fracture [11], [12]. Bone morphogenetic proteins (BMP) are members of the transforming growth factor (TGF-β) superfamily of signaling molecules and act as morphogens to regulate embryonic development [13], [14]. Evidence suggested BMP derived from mesenchymal cells and osteoblasts could exhibit chemotatic properties to stimulate differentiation of mesenchymal cells into osteogenic/chondrogenic lineage and increase expressions of alkaline phosphatase and osteocalcin [15], [16], [17]. In addition, BMP also affect bone remodeling through the regulation of osteoclast bone-resorbing activity [18]. BMP have been reported as having a role in the mechanical stimulation of fracture healing and chondrocyte differentiation [19], [20]. Four members of the BMP family, BMP-2, BMP-3, BMP-4, and BMP-7, have shown positive effects on facilitating fracture healing and bone formation [21], [22], [23], [24]. Thus, we postulated that BMP might be involved in the physical ESW promotion of fracture healing.

This study attempted to elucidate the effect of ESW treatment on cell proliferation and histomorphological changes in the healing of segmental defect. We also sought to clarify the spatial and temporal expression of BMP-2, BMP-3, BMP-4, and BMP-7 in the ESW promotion of bone regeneration of segmental femoral defects.

Section snippets

Segmental defect model

Three-month-old Sprague–Dawley rats (National Experimental Animals Production Center, Taipei, Taiwan) were used in this study. Animals were caged in pairs and maintained on rodent chow and water ad libitum. Rats were anesthetized by an intraperitoneal injection of pentobarbital sodium (50 mg/kg; Nembutal sodium, Abbott Laboratories, IL, USA). A four-hole AO/ASIF miniplate was positioned on the anteromedial femoral shaft after stripping of the periosteum. The proximal and distal holes were

Histomorphologic changes in segmental defect following ESW treatment

Histological observation demonstrated segmental defects with little or no new bone formation. The central regions of the defects were filled with fibrous tissue and skeletal muscle that had collapsed into the defect. The fractured cortex ends were surrounded with fibroblast, although no bone matrix was produced (Fig. 2). There was also no callus formation around the defects.

Major morphologic changes in segmental defects were observed 1 week after ESW treatment. Mesenchymal cells at the

Discussion

In this study, we demonstrate that ESW treatment of segmental femoral defects promoted cell proliferation and resulted in a cascade of bone regeneration that induced healing of the fracture gap. Interestingly, significant increases in BMP-2, BMP-3, BMP-4, and BMP-7 mRNA expression and intensive BMP immunoexpression of callus were noted during the fracture healing process. While a number of physical treatments have been employed for cartilage and bone repair, little has been done to define the

Acknowledgements

This work was supported in part by a grant from the National Health Research Institute, Taiwan [NHRI-EX91-9128EI (F.S.W.)]. The authors gratefully appreciate Dr. C.C. Huang, Department of Pathology, Chang Gung Memorial Hospital, Kaohsiung, Taiwan, for her assistance in histological evaluations.

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