Elsevier

Bone

Volume 44, Issue 2, February 2009, Pages 320-330
Bone

Stress fracture healing: Fatigue loading of the rat ulna induces upregulation in expression of osteogenic and angiogenic genes that mimic the intramembranous portion of fracture repair

https://doi.org/10.1016/j.bone.2008.09.010Get rights and content

Abstract

Woven bone is formed in response to fatigue-induced stress fractures and is associated with increased local angiogenesis. The molecular mechanisms that regulate this woven bone formation are unknown. Our objective was to measure the temporal and spatial expression of osteo- and angiogenic genes in woven bone formation in response to increasing levels of fatigue-induced damage. We used the rat forelimb compression model to produce four discrete levels of fatigue damage in the right ulna of 115 male Fischer rats. Rats were killed at 0 (1 h), 1, 3 and 7 days after loading. Using qRT-PCR, we quantified gene expression associated with osteogenesis (BMP2, Msx2, Runx2, Osx, BSP, Osc), cell proliferation (Hist4), and angiogenesis (VEGF, PECAM-1) from the central half of the ulna. The spatial distribution of BMP2, BSP and PCNA was assessed by immunohistochemistry or in situ hybridization in transverse histological sections 1, 4, and 7 mm distal to the ulnar mid-diaphysis. One hour after loading, BMP2 was significantly upregulated in neurovascular structures in the medial ulnar periosteum. Expression of angiogenic markers (VEGF, PECAM-1) increased significantly between Day 0 and 1 and, as with BMP2 expression, remained upregulated through Day 7. While Osx and BSP were upregulated on Day 1, the other osteogenic genes (Msx2, Runx2, Osx, BSP and Osc) were induced on Day 3 in association with the initiation of periosteal woven bone formation and continued through Day 7. The magnitude of osteogenic gene expression, particularly matrix genes (BSP, Osc) was significantly proportional the level of fatigue damage. The woven bone response to fatigue injury is remarkably similar to the “intramembranous” portion of fracture repair — rapid formation of periosteal woven bone characterized by early BMP2 expression, cell proliferation, and upregulation of osteogenic genes. We speculate that woven bone repair of fatigue damage may be an abbreviated fracture response without the requirement for endochondral repair. We conclude that bone fatigue repair is a process similar to intramembranous fracture repair characterized by increases in the expression of genes associated with angiogenesis, cell proliferation and osteoblastogenesis, and that the response from the local vasculature precedes the osteogenic response to fatigue loading.

Introduction

Woven bone forms during fracture healing, distraction osteogenesis and in response to stress fractures engendered by bone fatigue [1], [2]. Stress fractures occur commonly in people who engage in rigorous physical activities, such as military recruits and runners [3]. While a number of studies have explored the changes in gene expression associated with fracture healing [4], [5], [6], [7], [8], [9] and distraction osteogenesis [10], [11], [12], the molecular events that occur during woven bone formation in response to fatigue injury are unknown.

Fatigue loading of the rat ulna by cyclic compression of the forelimb results in the formation of stress fractures [13], [14], [15] and leads to a repair response characterized by rapid, non-endochondral woven bone formation [16]. We recently developed a protocol whereby the level of ulnar fatigue damage can be modulated [13]. Using this model we observed that the amount of woven bone formed [16] and the magnitude of associated vascular changes [17], [18] are proportional to the level of fatigue damage. There is also a strong spatial correspondence between the formation of woven bone and the location of fatigue-induced bone damage. Fatigue cracks form within the medial ulnar cortex [14], [15] and are centered near the mid-diaphysis [13]. Maximal woven bone formation occurs at the same location, and diminishes equally in the proximal and distal directions [16], [19].

In the “intramembranous” period of fracture repair, within the first seven days after fracture, there is a similar symmetric formation of woven bone on either side of the fracture site [20]. Contributions to the fracture repair process are thought to derive from four main areas: the periosteum, the original bone cortex, external soft tissues, and the bone marrow, with much of the chondrogenic response derived from the marrow component and surrounding soft tissues [20]. The early intramembranous response is also thought to be initiated by endothelial cells within the bone marrow that take on an osteoblastic phenotype within 24 h after fracture [20], [21]. Periosteal stress fractures resulting from fatigue damage do not typically involve direct contact with the endosteum. Nevertheless, the woven bone response to fatigue damage and the intramembranous portion of fracture repair share morphological and temporal similarities.

The formation of new blood vessels (vasculogenesis or angiogenesis) plays an important role during osteogenesis and bone healing [22]. In fracture repair [23], [24] and distraction osteogenesis [11], [25], [26], vascular changes precede bone formation. In the rat ulnar fatigue model, the spatial pattern of maximal vascular changes along the ulna is identical to the pattern of woven bone formation [18]. Moreover, we observed increases in vessel size in the periosteum on day 1 after fatigue loading [17] and measured significant increases in vessel size and number on day 3 [18], whereas significant increases in woven bone area were not evident until after day 3 [17], [18]. We therefore expect that the upregulation of genes associated with angiogenesis precedes the upregulation of genes associated with osteogenesis following fatigue loading.

Our objective was to extend prior histological descriptions of stress fracture healing by characterizing the temporal–spatial pattern of the expression of bone formation and angiogenic genes. We used quantitative real-time polymerase chain reaction (qPCR) along with immunohistochemistry and in situ hybridization to assess gene expression following fatigue loading and creation of an ulnar stress fracture in rats.

Section snippets

Experimental design

Male Fischer/NHsd rats (N = 115; 4.5–5.5 months old; 339 ± 29 g; Harlan) were assigned randomly to one of four loading groups according to the level of imposed fatigue damage and one of four survival time points. As described below, right forelimbs were loaded cyclically to a prescribed displacement level corresponding to 30, 45, 65 or 85% of the average displacement to fracture (2.0 ± 0.02 mm). We have shown previously that these displacement levels correspond to four discrete levels of ulnar

Day 0: BMP2 is rapidly upregulated in extra-osseous structures of fatigue-loaded ulnae

Of the nine genes reflecting osteogenic-angiogenic coupling potential in this model, only BMP2 was consistently and significantly upregulated at all time points with all extents of loading. At Day 0, BMP2 expression was significantly elevated, up by an average of 3-fold in loaded compared to non-loaded control ulnae in all displacement groups (Table 3). VEGF and PECAM-1 were also upregulated in loaded versus control ulnae in the 45, 65, and 85% groups. Other genes did not show a significant

Discussion

Our objective was to characterize the effects of increasing fatigue damage on the expression of osteogenic and angiogenic genes using an established model of fatigue loading that creates a rat ulnar stress fracture [14], [15]. We recently documented that our loading protocol induces woven bone formation at the ulnar mid-diaphysis in proportion to the level of fatigue damage, leading to recovery of ulnar strength two weeks after loading [16]. While a number of studies have explored molecular

Acknowledgments

Funding was provided by grant AR050211 from the NIH/NIAMS. We wish to thank Stefan Rothermich, Carl Franz and Dr. Deborah Novack for their assistance with histology.

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