The Effect of Mechanical Stimulation on Bone Fracture Healing: Changes in Callus Morphology and Mesenchymal Stem Cell Homing.

Abstract

This dissertation examines how the timing of an applied strain influences callus morphology during bone fracture repair, and how this load effects the migration of systemically delivered mesenchymal stem cells (MSCs) to the fracture site. Six-month-old, male, Sprague-Dawley rats underwent a 2mm segmental osteotomy in the mid-diaphysis of each femur. A two-piece external fixator with locking plate was fixed to each femur, an osteotomy created with an oscillating saw, and the surrounding tissues closed. Systemic injections of green fluorescent protein (GFP) labeled MSCs were performed via the tail vein on the first loading day. Axial mechanical stimulation occurred for five consecutive days at 0, 3, 10, or 24 days post-operative at a magnitude of ±8% strain and a rate of 0.313 Hz for 510 cycles. Rats were euthanized 10, 24, or 48 days post-op. Through a micro-CT analysis, mineralization was found to be increased in animals that underwent the applied displacement at day ten when compared to the other groups. Histology showed that by day 48, these animals had almost no cartilage remaining, indicating advanced remodeling in comparison to other groups. The animals stimulated at day three showed the opposite trends, with less mineralization and larger, less remodeled calluses by day 48. Planar gamma imaging of 111indium tagged MSCs showed that injected cells migrate from the lungs and liver, and a certain percentage of them are available to the limbs. Axial displacement created some difference in MSC migration to the limbs, but these disappeared by day three. As analyzed by immunohistochemistry, there were significant numbers of MSCs in the marrow spaces by day 48, and a qualitative analysis suggests that stimulation may encourage the cell to preferentially migrate. A polymerase chain reaction (PCR) array examined the genetic expression of 80 genes in the fracture gap tissue implicated in migration of MSC migration. It showed that the expression of many different genetic factors is influenced by mechanical stimulation. This effect also appears to not just be localized, but can be seen systemically as well. Overall, the timing of displacement had a significant effect on fracture healing, both locally and systemically.