Temporal pattern of gene expression and histology of stress fracture healing

Abstract

Loading of the rat ulna is an ideal model to examine stress fracture healing. The aim of this study was to undertake a detailed examination of the histology, histomorphometry and gene expression of the healing and remodelling process initiated by fatigue loading of the rat ulna. Ulnae were harvested 1, 2, 4, 6, 8, and 10 weeks following creation of a stress fracture. Stress fracture healing involved direct remodelling that progressed along the fracture line as well as woven bone proliferation at the site of the fracture. Histomorphometry demonstrated rapid progression of basic multicellular units from 1 to 4 weeks with significant slowing down of healing by 10 weeks after loading. Quantitative PCR was performed at 4 hours, 24 hours, 4 days, 7 days, and 14 days after loading. Gene expression was compared to an unloaded control group. At 4 hours after fracture, there was a marked 220-fold increase (P<0.0001) in expression of IL-6. There were also prominent peak increases in mRNA expression for OPG, COX-2, and VEGF (all P<0.0001). At 24 hours, there was a peak increase in mRNA expression for IL-11 (73-fold increase, P<0.0001). At 4 days, there was a significant increase in mRNA expression for Bcl-2, COX-1, IGF-1, OPN, and SDF-1. At 7 days, there was significantly increased mRNA expression of RANKL and OPN. Prominent, upregulation of COX-2, VEGF, OPG, SDF-1, BMP-2, and SOST prior to peak expression of RANKL indicates the importance of these factors in mediating directed remodelling of the fracture line. Dramatic, early upregulation of IL-6 and IL-11 demonstrate their central role in initiating signalling events for remodelling and stress fracture healing.

Atypical subtrochanteric and diaphyseal femoral fractures: Report of a task force of the american society for bone and mineral Research

“…(69) Still, primary studies in nonskeletal tissues suggest that angiogenesis indeed may be reduced by BPs over and above the normal reduction that would occur because of the absence of effective osteoclastic tunneling. (70) Interestingly, in a rat model of stress fracture, there is upregulation of vascular endothelial growth factor (VEGF) mRNA within 1 to 4 hours of initiation of the stress fracture (71,72) and upregulation of osteogenic genes in the cambium layer of the periosteum within 3 days. Early upregulation of interleukin 6 (IL-6) and IL-11 suggest the importance of remodeling in stress fracture healing.…”

Bisphosphonate treatment delays stress fracture remodeling in the rat ulna

“…15 Loading was performed in a custom designed loading device which has previously been described in detail. 20 To determine displacement in the limb during loading, the loading device was attached to a linear vertical displacement transducer (LVDT) (made in the School of Biomedical Sciences electrical workshop) connected to a maclab (AD Instruments, Colorado Springs, Colorado) and monitored using Chart v5.4 (AD Instruments).…”

“…This section was the point where the fracture line was half way between the medial cortical margin and the medullary cavity of the bone in transverse section. 15 Toluidine blue staining provided excellent cellular and structural detail. Areas of woven bone, areas of osteoclastic resorption and cement lines between the original and newly formed intracortical bone could be clearly distinguished and measured (Fig.…”

“…3,12,13 In animal models, stress fractures heal by direct remodeling along the fracture line. 14,15 Treatment with bisphosphonates may therefore have a negative effect on stress fracture healing when compared to complete fractures. For example, alendronate reduced the number of resorption spaces observed along stress fracture lines induced by ulnar loading in the rat.…”

“…15 Stress fracture healing in this model consists of two distinct healing components; the rapid woven bone response on the periosteal surface and direct remodeling, in the form of numerous basic multicellular units (BMUs) progressing along the fracture line. 14,15,17,18 The aim of this study was to determine the effect of long-term daily risedronate treatment on the healing of stress fractures in the rat ulna. High and low doses were selected to determine if a dose-response existed, and oral treatment was used to represent a clinical protocol.…”