Shock wave application enhances pertussis toxin protein-sensitive bone formation of segmental femoral defect in rats.
Extracorporeal shock waves (ESWs) elicit a dose-dependent effect on the healing of segmental femoral defects in rats. After ESW treatment, the segmental defect underwent progressive mesenchymal aggregation, endochondral ossification, and hard callus formation. Along with the intensive bone formation, there was a persistent increase in TGF-beta1 and BMP-2 expression. Pretreatment with pertussis toxin reduced ESW-promoted callus formation and gap healing, which presumably suggests that Gi proteins mediate osteogenic signaling.


BACKGROUND
Extracorporeal shock waves (ESWs) have previously been used to promote bone repair. In our previous report, we found that ESWs promoted osteogenic differentiation of mesenchymal cells through membrane perturbation and activation of Ras protein. In this report, we show that ESWs elicit a dose-dependent effect on the healing of segmental defects and that Gi proteins play an important role in mediating ESW stimulation.


METHODS
Rats with segmental femoral defects were subjected to ESW treatment at different energy flux densities (EFD) and impulses. Bone mass (mineral density and calcium content), osteogenic activities (bone alkaline phosphatase activity and osteocalcin content), and immunohistochemistry were assessed.


RESULTS
An optimal ESW energy (500 impulses at 0.16 mJ/mm2 EFD) stimulated complete bone healing without complications. ESW-augmented healing was characterized by significant increases (p < 0.01) in callus size, bone mineral density, and bone tissue formation. With exposure to ESW, alkaline phosphatase activity and osteocalcin production in calluses were found to be significantly enhanced (p < 0.05). After ESW treatment, the histological changes we noted included progressive mesenchymal aggregation, endochondral ossification, and hard callus formation. Intensive bone formation was associated with a persistent increase in transforming growth factor-beta 1 (TGF-beta1) and bone morphogenetic protein-2 (BMP-2) expression, suggesting both growth factors were active in ESW-promoted bone formation. We also found that pertussis toxin, an inhibitor of membrane-bound Gi proteins, significantly reduced (p < 0.01) ESW promotion of callus formation and fracture healing.


CONCLUSIONS
ESW treatments enhanced bone formation and the healing of segmental femoral defects in rats. It also seems likely that TGF-beta1 and BMP-2 are important osteogenic factors for ESW promotion of fracture healing, presumably through Gi protein-mediated osteogenic signaling.
Chen
Yeung-Jen
YJ
Department of Orthopedic Trauma, Chang Gung University, Linkou, Taiwan.
Kuo
Yur-Ren
YR
Yang
Kuender D
KD
Wang
Ching-Jen
CJ
Huang
Hue-Chen
HC
Wang
Feng-Sheng
FS
D015502:Absorptiometry, Photon; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015519:Bone Density / Q000502:physiology; D001846:Bone Development / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D004597:Electroshock; D010010:Osteoclasts / Q000187:drug effects / Q000502:physiology*; D037342:Pertussis Toxin / Q000494:pharmacology*; D051381:Rats; D016212:Transforming Growth Factor beta / Q000502:physiology
C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D016212:Transforming Growth Factor beta; D037342:Pertussis Toxin; D000469:Alkaline Phosphatase; D002118:Calcium