Inhibiting IGF1 Activity in the Proximal Tibial Growth Plate Attenuates the Bone‐Lengthening Effects of Temperature in Hindlimbs of Growing Mice

Limb length inequality in children can lead to conditions such as scoliosis, back pain and osteoarthritis. With limited treatment options, we sought to develop approaches for modulating limb lengthening without invasive surgeries. Bone elongation in cartilaginous growth plates is regulated by many mechanisms involving local and systemic growth factors, which can be altered by environmental inputs such as temperature. We previously developed a once daily unilateral heating regimen to increase limb length using targeted heat exposure on one side of the body of growing mice. Using this model, we were able to demonstrate an increase in tibial elongation rate of over 12% on the heat‐treated side. OBJECTIVE Here we aimed to determine whether insulin‐like growth factor 1 (IGF1) is involved in the heat‐enhanced growth effect since limb elongation is IGF1 dependent. We HYPOTHESIZE that unilateral exposure to warm temperature augments actions of IGF1 in the growth plate to enhance bone length. To test this, we coupled our limb‐heating model with an IGF1 peptide analog (JB1, Sigma) that blocks downstream activity of IGF1. If the lengthening effect of temperature is IGF1‐dependent as hypothesized, we expect to find reduced limb growth when the blocking drug is administered with heat. METHODS Each morning for 7 days, 3‐week old female C57BL/6 mice (N=6 per group) were injected with saline, IGF1 (2.5mg/kg SQ), or the IGF1 blocking drug JB1 (2.5mg/kg) one‐hour prior to unilateral heat‐treatment (40C for 40 min/day). Proximal tibial growth plates were then collected and femora were dissected, cleaned, and measured. Tibiae were decalcified and paraffin embedded prior to immunohistochemical staining using a phosphorylated Akt (pAkt) antibody to assess IGF1 signal activation. Statistical significance (p<0.05) was determined using one‐way ANOVA and paired t‐tests. RESULTS Heat‐treated femora in both saline and IGF1‐injected groups were nearly 1.7% longer than their non‐treated contralateral side. There were no significant left‐right differences in femoral length in the IGF1 blocking drug (JB1) group (p=0.8). Histological analyses revealed that JB1‐injected mice had significantly shorter growth plates than either saline or IGF1‐injected mice with marked differences in proliferative and hypertrophic zones. Left‐right growth plate comparisons showed reduction in proliferative zone, and enlargement in hypertrophic zone heights on the heat‐treated side of both saline and IGF1‐injected groups, while the opposite was seen inJB1‐injected mice. Immunostaining showed a trend of increased pAkt expression on the heat‐treated sides of saline and IGF1 groups, suggesting an IGF1‐driven increase in growth rate. This growth acceleration was blocked in mice that received JB1. We are currently quantifying PCNA expression to compare cell proliferation rates. CONCLUSION We conclude that blocking IGF1 activity in the proximal tibial growth plate attenuates the bone lengthening effects of temperature in hindlimbs of growing mice. Results are relevant for elucidating mechanisms of heat‐enhanced bone elongation and for developing strategies to combat a range of limb lengthening disorders. Support or Funding Information Supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (1R15AR067451‐01)