Effects of mechanical loading on cortical defect repair using a novel mechanobiological model of bone healing

Highlights

• A novel murine mechanobiological model is described.

• Effects of loading during distinct stages of cortical defect repair were examined.

• Early loading disrupts the injury site and activates cartilage formation.

• Intermediate loading enhances bone and cartilage formation.

• Late loading results in an enlarged woven bone regenerate.

Abstract

Mechanical loading is an important aspect of post-surgical fracture care. The timing of load application relative to the injury event may differentially regulate repair depending on the stage of healing. Here, we used a novel mechanobiological model of cortical defect repair that offers several advantages including its technical simplicity and spatially confined repair program, making effects of both physical and biological interventions more easily assessed. Using this model, we showed that daily loading (5 N peak load, 2 Hz, 60 cycles, 4 consecutive days) during hematoma consolidation and inflammation disrupted the injury site and activated cartilage formation on the periosteal surface adjacent to the defect. We also showed that daily loading during the matrix deposition phase enhanced both bone and cartilage formation at the defect site, while loading during the remodeling phase resulted in an enlarged woven bone regenerate. All loading regimens resulted in abundant cellular proliferation throughout the regenerate and fibrous tissue formation directly above the defect demonstrating that all phases of cortical defect healing are sensitive to physical stimulation. Stress was concentrated at the edges of the defect during exogenous loading, and finite element (FE)-modeled longitudinal strain (ε_zz) values along the anterior and posterior borders of the defect (~ 2200 με) was an order of magnitude larger than strain values on the proximal and distal borders (~ 50–100 με). It is concluded that loading during the early stages of repair may impede stabilization of the injury site important for early bone matrix deposition, whereas loading while matrix deposition and remodeling are ongoing may enhance stabilization through the formation of additional cartilage and bone.

Introduction

Bone repair is a highly organized and controlled process involving specialized cells and signaling molecules that are precisely coordinated in time and space. The bone repair program is acutely sensitive to mechanical stimulation [1], [2], [3], [4], [5], [6], and early weight-bearing is an important aspect of orthopaedic post-surgical care [7]. How mechanical signals are sensed and integrated to regulate healing is incompletely understood. Characterizing the mechanical environment during bone repair and understanding its effects on gene expression, cell behavior, and tissue level properties is essential for the development of novel therapeutic treatments, both physical and pharmaceutical, for complex orthopaedic injuries in weight-bearing bones.

Previous studies using osteotomy and segmental defect models examined the influence of mechanical loading on healing [8], [9], [10], [11], [12], [13], [14]. The effects of load magnitude and latency period on healing were investigated using a pinned tibial osteotomy model [8]. Compressive axial loading (100 cycles/day, 1 Hz, 5 days per week for 2 weeks at 0.5 N, 1 N, and 2 N peak load) was applied across the flexed knee and ankle immediately after fracture or after a 4-day delay, which coincided with the hematoma and inflammation stages. Loading applied immediately after fracture during the acute injury stage inhibited callus formation regardless of load magnitude, resulting in reduced callus strength and stiffness compared to non-loaded controls. Of all combinations of load magnitude and latency period, only the 0.5 N load applied at 4 days post-fracture resulted in a stronger callus relative to non-loaded controls.

Additional studies have shown that femoral segmental defects subjected to daily cyclic bending (900 cycles, 1 Hz, 15 min/day for 5 consecutive days per week for 1, 2 or 4 weeks) beginning on post-surgical day 10, which coincided with a provisional matrix scaffold, led to formation of pseudarthrosis with enhanced cartilage formation [9], [10], increased expression of cartilage-related genes COL2A1 and COL10A1 [9] and mechanically-activated genes FAK and RhoA [11], and decreased expression of bone-related genes BMP-4, -6, and -7 [9]. In other studies, the effects of continuous compressive ambulatory loading on repair of a critical sized femoral defect were investigated [12], [13]. Defects were treated with rhBMP-2 and fixed with either stiff or adjustable locking plates, which when unlocked served as compliant fixation. Adjustable plates were unlocked at either day 0 (acute injury phase) or 4 weeks post-fracture when low-density bone was present in the defect [14]. Compliant fixation beginning on day 0 resulted in significantly reduced vascularization and bone formation compared to stiff plate controls. In contrast, compliant loading beginning after a 4-week delay led to enhanced bone and cartilage formation and stimulated vascular remodeling [12], [13]. Together these results suggest that loading initiated during the acute injury phase (day 0) disrupts stabilization of the injury site and inhibits bone formation, whereas loading initiated after bone matrix has been deposited enhances both bone and cartilage formation.

While these studies reveal effects of long-term habitual loading on healing, it is less clear how short-term acute loading confined to distinct stages of repair affects cellular activity, tissue formation and overall healing. Furthermore, the previously described models rely on fixation hardware (e.g., intramedullary pin, plates, external fixator) for stabilization, which can exert secondary effects on healing including inflammation at the site of implantation [15], infection [16], and disruption of the marrow cavity with intramedullary pinning, all of which may mask direct effects of mechanical stimulation on repair. Additionally, any inconsistencies in surgical implant placement may introduce variability in the amount of micromotion at the injury site across samples [12], [17], [18], [19].

The aim of this study was to determine the effects of short-term exogenous mechanical loading during early (inflammation), intermediate (matrix deposition), and late (remodeling) stages of repair on cortical defect healing. Here we utilize a self-stabilizing monocortical defect model [20], [21] that undergoes a well-defined intramembranous repair program and that is amenable to application of precisely controlled axial compressive loading regimens [22], [23], [24], [25] at defined stages of healing. We chose this intramembranous repair model because any formation of cartilage and fibrous tissue could be attributed to the applied load, and the absence of radio-opaque fixation hardware: (1) eliminated potential secondary effects of hardware, (2) permitted in vivo longitudinal microCT scanning, and (3) facilitated development of FE models for estimation of load-induced mechanical strain at the injury site. We hypothesized that loading during distinct stages of repair would differentially influence cellular activity, tissue formation and healing, and if supported, may aid in the development of biophysical rehabilitative strategies to enhance fracture repair.