SMAD EXPRESSION IN FRACTURE HEALING

Abstract

Fracture healing involves many local and systemic regulatory factors. Progress in identifying signaling events downstream has been made with the discovery of a novel family of proteins, the Smad, as TGF-ß/activins/BMPs signal transducers. Smads are the vertebrate homologs of Mad (Mothers against decapentaplegic) gene from Drosophila and Sma genes from Caenorhabditis elegans. Smad-1, -2, -3, -5, -8 and -9 belong to the receptor-regulated class (R-Smad) which are activated by the TGF-ß type I and II receptors, forming heteromers with the common-mediator class (Co-Smad): Smad-4. Smad-6 and -7 (Anti-Smad) perform a negative regulatory or balancing role. Smad-2 and -3 regulate TGF-ß/activin effects, whilst Smad-1 and -5 work with BMPs. This study investigated the expression and localization of Smad proteins (Smad 1–6) and BMP-4 and -7 during fracture healing.

Eighteen 3-month old female CD-COB rats were used. A standard closed fracture was made in the mid-shaft of right femur using a 3-point bending device. The left limb served as the non-fracture control. The rats were divided into 3 groups (6 per group) and sacrificed at day 3, 10 and 28 after fracture. The femurs were harvested, fixed in buffered formalin for 48 hours and decalcified with 10% formic acid-formalin solution. The decalcified tissues were embedded in paraffin and 5μm sections were cut onto silane-coated slides. Representative slides from each block were stained with routine haematoxylin and eosin (H& E). Sections were cut for immunohistochemistry for protein marker expression by a standard procedure for Smads and BMP 4 and 7. Sections were viewed and analysed by colour video image analysis using a 40x objective, a 10x eyepiece, and a fixed frame of 128 × 128 pixels (49152.0 μm²). Ten fields per slide were examined.

Smad proteins (Smads 1, 4, and 6) were expressed during the early stages (day 3) of fracture healing by bone marrow stromal cells, osteoblasts, fibroblasts and chondrocytes located in the intramembranous and endochondral ossification regions around the fracture site. Differential expressions of individual Smads, particularly Smad 1 and Smad 6, at different time-points (Smad-1 was higher than Smad-6 at day 3, whilst Smad-6 was much higher than Smad-1 at day 10) suggest that Smad proteins are not simply BMP signal transducers. Smads may also be responsible for up- and/or down-regulation of transcriptional events during the intramembranous and endochondral ossification. Smad-4, a Co-SMAD, expression newly formed bone and cartilage suggests an additional function beyond the signal transduction in rat fracture healing. BMP-4 and BMP-7 were highly expressed at day 3 and 10. BMP-7 expression was greater than BMP-4 at day 3 but switched by day 10 (BMP-4 > BMP-7). Smads represent a new level where specific therapeutic strategies can be targeted considering the interactions with a number of BMPs.