Early callus of fractured rib of rat contracts and relaxes ex vivo

Purpose. Wound contraction is an essential process in early soft-tissue repair, yet contraction of callus in fracture repair has not been investigated previously. Fracture callus consists of several cell types, many of which may have the capacity to contract. Accordingly, the purpose of the present study was to (i) determine whether early soft fracture calluses contract and relax ex vivo and (ii) identify and locate the contractile protein, alpha smooth muscle actin (αSMA) in callus. Methods. One non-weight-bearing rib was fractured in adult male rats under anaesthesia and 10 calluses were removed 5, 7 and 9 days later for examination. Force production by calluses was measured using a sensitive force transducer when callus preparations were immersed sequentially in solutions known to either contract or relax smooth muscle preparations. Calluses and unfractured rib were analysed for the presence of αSMA using Western Blot and immunohistochemical techniques. Results. When immersed in normal Krebs–Henseleit solution (K–H; pH 7.4, 22 °C) 7 callus preparations contracted and 3 relaxed. The force response was phasic (3 calluses) or tonic (7 calluses). Subsequent immersion in Ca 2+-free K–H resulted in no change in force in 4 calluses, a decrease in force (relaxation) in 3 calluses, and an increase in force (contraction) in 2 calluses when compared to the force in the preceding solution (K–H). The final incubation in a solution having a high [K +] (64 mM) partially relaxed 6 calluses, contracted 3 and produced no change in force in 1 callus compared to the final force of the callus in the Ca 2+-free solution. Collagen (in the form of rat Achilles tendon), the major structural protein in soft fracture callus, relaxed in K–H and continued to relax during exposure to Ca 2+-free K–H and to solutions having a high [K +]. Western Blot and immunohistochemical studies detected the presence of αSMA in calluses and (in particular) in osteoprogenitor cells of fibrous callus respectively, as well as its absence from unfractured rib. Conclusions. (i) Early, soft fracture callus is capable of contracting and relaxing, (ii) the responses of callus to K–H, Ca 2+-free and high [K +] solutions are distinctly different from the responses of smooth muscle preparations reported in the literature, (iii) the cell types in callus, particularly osteoprogenitor cells in uncalcified, collagenous matrix, have an essential contractile protein, αSMA, to support the observed contraction and re