Distinct functionalities of bone morphogenetic protein antagonists during fracture healing in mice - PubMed (original) (raw)

Distinct functionalities of bone morphogenetic protein antagonists during fracture healing in mice

Daniel B Dean et al. J Anat. 2010 May.

Abstract

The bone morphogenetic protein (BMP) family of growth factors plays critical roles in bone formation. BMPs are regulated at multiple levels by various BMP antagonists. This study investigated how BMP antagonists are integrated into the cascade of events of bone formation during fracture healing. Forty mice underwent a controlled femur fracture; tissue samples at the fracture site were harvested at days 1, 3, 7, 14 and 21 after fracture, for quantification of the expression of BMPs and BMP antagonists. During fracture healing, BMP-2, -4 and -7 were up-regulated, but BMPR-1A and BMPR-2 showed reduced expression after day 14. Among BMP antagonists, the expressions of PRDC, SOST, Smad7, GREM1 and CERBERUS were generally down-regulated during fracture healing. In contrast, Noggin was significantly up-regulated in the first week after fracture; 7 days after fracture, other BMP antagonists, including DAN, CHRD, Smad6 and BAMBI, also showed significantly increased expression. In conclusion, this study indicates that BMP antagonists can be divided into two functional groups in relation to fracture healing: (1) those whose suppression may be essential for the initiation of osteogenesis; (2) those that are upregulated and may function in the remodeling of newly formed bone.

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Figures

Fig. 1

Fig. 1

Histology of bone healing. Day 1: blood clot formed at the fracture end. Day 3: cell density increased in the clot as results of cell proliferation and local inflammation. Day 7: fibroblasts condensed at the fracture end. Day 14: callus formed at the fracture site with mixed tissues of fibrous, cartilaginous and bone. Day 14a: enlarged area of the cartilaginous tissue in the callus. Day 21: callus matured with mostly bone and bone marrow formed in the bone (Day 21a). Note: hematoxylin and eosin staining.

Fig. 2

Fig. 2

Quantitative RT-PCR. Gene expression in the controls, i.e. normal non-fractured bone, is defined as 1. _Y_-axis shows the fold number of the expression of genes investigated relative to the controls ((ΔΔCt). (A) There is a significant upregulation of BMP-2 and -7 at day 7, and the upregulation of BMP-7 continued into week 3. The expression of BMP-4 is above the control throughout the fracture-healing period, but reaches significant levels only after week 2. The expression of BMPR-1A and BMPR-2 in the callus is insignificantly different from the controls, except at day 14. (B) Among the BMP antagonists, PRDC and GREM1 in callus are expressed less than half of that in normal non-fractured bone. CERBERUS and Smad7 are expressed at a lower than normal level at all time-points, but this is only significant at day 14. The expression of SOST is downregulated during fracture healing, except at day 7. (C) DAN, CHRD, noggin, BAMBI, and Smad6 are upregulated during fracture healing. Most of these show a trend of a gradual increase of expression, but noggin is upregulated only during the first week of fracture. The greatest upregulation is nearly 50-fold of DAN at day 21. *Indicates where P < 0.05 when the gene expression is compared between the fracture tissue and normal bone.

Fig. 3

Fig. 3

Immunohistochemistry for DAN. At week 1, the fractured bone is not stained with DAN (A). In weeks 2 (B) and 3 (C), DAN is strongly stained in the newly formed woven bone, but not in the mature lamellar cortical bone. DAN is stained with Texas Red, red. Nuclear is counterstained with DAPI, blue. C: cortical bone; W: woven bone; bar = 100μm.

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