Introduction and Aims: Although skeletal muscles have remarkable potential for adaptation, the amount of muscle length increase during gradual limb lengthening is always less than the amount of bone lengthening. The purpose of this study was to analyse gene expression in skeletal muscle undergoing adaptation to limb lengthening.

Method: Ten adult goats were randomly divided into two groups of five animals. Group 1 underwent 20% (43–47mm) standard Ilizarov tibial lengthening and group 2 served as un-operated control. Muscle tissues from proximal myotendenous junctions of Peroneus Longus were harvested from the lengthened limb in the distraction group and corresponding limb in the control group and immediately snap frozen in liquid nitrogen. To screen for genes potentially associated with sarcomerogenesis, microarray technology was employed. Biotin labeled cRNA was hybridised to Affymetrix HU133A GeneChips, containing 22,284 gene transcripts. All created data files were analysed using computer software GeneSpring 5.0.

Results: In both groups, 5092 (23%) gene transcripts flagged present. Thirty-two of these transcripts were differentially expressed between distracted and control groups (p < 0.05). Represented by these transcripts were 12 known and three unknown genes, which were up-regulated in lengthened muscles by more than 2.0 fold. The substantially up-regulated genes identified were MYOZ2 (myozenin 2), MYL4 (embryonic myosin alkali light chain), MYL6 (myosin light polypeptide 6), CRYAB (crystalline), PFN2 (profiling 2), ARPP-19 (cyclic AMP phosphorprotein), TUBB2 (tubulin beta 2), PPP1R12 (protein phosphatase 1), RCOR (REST corepressor), LIM (LIM protein), FN1 (fibronectin 1), ACTC (alpha-actin), and hypothetical protein FLJ10111. Among the genes found to be up-regulated are genes involved in the myogenesis pathway. Myozenin 2 gene is associated with the signalling and activity of Calcineurin/Calsarcin that plays a significant role in muscle cell proliferation and myofiber type differentiation. Crystallin gene may be involved in promoting muscle survival during differentiation. The functionality of the remaining genes range from cytoskeletal organisation (TUBB2), cyto-skeletal structure (PFN2, MYL4, MYL6), cell adhesion and motility (FN1), muscle development and differentiation (FHL1 and LIM), intercellular adhesion and intermediate filament organisation (PNN), muscle contraction and relaxation (PPP1R12A), neuronal-specific gene silencing (RCOR), and PKA-dependent intracellular messaging (ARPP-19).

Conclusion: The findings suggest that tension stress observed during gradual limb lengthening using standard Ilizarov distraction protocol activates expression of genes involved in skeletal muscle growth, differentiation, and neogenesis. On-going studies involving immunohistochemistry, RT-PCR, and in situ hybridisation to confirm cellular localisation of up-regulated genes are underway.

Introduction and Aims: With a great progress in bone regeneration, muscle is currently regarded as a largest limiting factor for successful limb lengthening leading to joint contractures and fractures of distraction regenerate. The purpose of this study was to evaluate muscle architectural changes and potential mechanisms of joint contractures during limb lengthening.

Method: Nine mature goats underwent 20% unilateral tibial lengthening (0.25 mm x 3/day) and were sacrificed immediately upon completion of distraction. With the stifle (knee) and hock (ankle) joints fixed at similar angles, both limbs were disarticulated at the hip joint and submerged into 10% buffered formalin. Following tissue fixation, all tibial muscles were sequentially dissected and changes in muscle origin-to-incretion length, belly length, tendon length, myofibers length, and sarcomere length were analysed relative to the muscle measurements on the contralateral limb and bone lengthening. Muscle fiber length was assessed under stereoscopic magnification and sarcomere analysis was performed using laser diffraction.

Results: Thirteen muscles were identified for each limb. Anterior compartment consisted of two longitudinal and four pennate muscles, whereas posterior compartment had one longitudinal and six pennate muscles. Origin-to-insertion length measurements showed disproportion between the amounts of muscle and bone length increase with muscle-to-bone lengthening ratio ranging from 0.2 to 1.0. When assessed separately, muscle belly stretched more substantially (range, 11–24%) than muscle tendon (range, 3–14%). Longitudinal muscles showed better compliance to limb lengthening than pennate muscles. Origin-to-insertion, muscle belly, and tendon length increase for longitudinal muscles averaged 15%, 21%, and 11%, respectively, whereas for pennate muscles these parameters averaged 10%, 15%, and 6%, respectively. Although anterior pennate muscles showed higher proportion of muscle length increase than posterior pennate muscles, this difference was not significant. Lengthening of muscle fibers varied greatly, ranging from 0% to 88%. Fiber length of posterior muscles increased tremendously (average, 42%). This was associated with comparable increase in sarcomere length (average, 39%). Anterior muscles showed only 10% lengthening of the fibers. However, 12% reduction in sarcomere length indicated addition of new sarcomeres in series to accommodate increase in fiber length.

Conclusion: Different response of anterior and posterior muscles to distraction contributed greatly to the development of joint contractures. Posterior tibial muscles were predominantly pennate, larger in volume, and thus showed higher resistance to lengthening. Moreover, posterior muscle fibers incurred lengthening by sarcomere stretching, whereas anterior muscle fibers showed evidence of neosarcomerogenesis.