Defective TGF beta signaling in bisphosphonates associated atypical femoral fracture

Abstract

INTRODUCTION: Bisphosphonates (BPs) are potent anti-resorptive agents and their therapeutic use in osteoporosis treatment and fracture prevention has been well established. However, studies investigating the association of atypical femoral fractures (AFF) and use of BPs are still inconclusive due to lack of scientific evidence. Transforming growth factor-beta (TGF-beta) is abundant in bone matrix which plays critical roles in bone remodeling, migration of bone mesenchymal stem cells to resorptive sites, osteoblastic-osteoclastic coupling in bone formation and resorption. Increase of TGF-beta1 has also been shown as the etiology of disease which exhibits diaphyseal dysplasia. PURPOSE OF STUDY: Purpose of this study was to compare expression of TGF-beta in AFF with normal control and to determine TGF-beta as possible cause of AFF. METHODS: Bone marrow supernatant was collected for active TGF-beta levels. Bone marrow stromal cells from AFF patient and control were obtained. Pluripotency of stromal cells had been demonstrated by adipogenic, chondrogenic and osteogenic differentiations. These cells were differentiated to osteoblasts and osteoclasts for in-vitro co-culture with bovine femoral cortical bone discs. Osteogenic differentiation rate was reported by ALP assay. The difference in rate of cells metabolic activities was evaluated by MTT assay. Gene and protein expressions of TGF-beta were reported by q-RT-PCR and cytokine ELISA. After initial comparison of findings between AFF and control samples, further culturing of control sample with alendronate and in-vito treatment of AFF sample by TGF-beta 1 were performed. RESULTS: In initial comparison, active TGF-beta 1 was detected as significantly decreased in bone marrow supernatant of AFF group by cytokine ELISA; higher osteogenic differentiation rate was found in AFF samples in contrary to low clinical serum ALP level in BP-treated patients. The rate of osteoblast metabolic activity was found significantly different in co-cultures with variety of spatial arrangement. Gene expressions of TGF-beta 1 and TGF-beta R1 were found down-regulated; TGF-beta 2 found up-regulated and TGF-beta R2 found down-regulated in AFF sample. ELISA results showed free release of TGF-beta 1 in medium is lower in AFF. In the follow-up culture of control sample with alendronate exhibited change of all ALP, MTT, q-RT-PCR and ELISA findings towards trends of AFF results. Whereas, in-vitro treatment of AFF sample by TGF-beta 1 demonstrated restorative effect towards control level. CONCLUSION: TGF-beta signaling pathway was found different in AFF patients from normal control. Further culture studies recommended causal relationship of defective TGF-beta signaling and BPs associated AFF. BPs affected bone marrow stromal cells which led to this defective TGF-beta signaling.