Mechanical cues are known to regulate tissue differentiation during skeletal healing. Quantitative characterization of this mechano-regulatory effect has great therapeutic potential. This study tested an existing theory that shear strain and interstitial fluid flow govern skeletal tissue differentiation by applying this theory to a scenario in which a bending motion applied to a healing transverse osteotomy results in cartilage, rather than bone, formation. A 3-D finite element mesh was created from micro-computed tomography images of a bending-stimulated callus and was used to estimate the mechanical conditions present in the callus during the mechanical stimulation. Predictions regarding the patterns of tissues-cartilage, fibrous tissue, and bone-that formed were made based on the distributions of fluid velocity and octahedral shear strain. These predictions were compared to histological sections obtained from a previous study. The mechano-regulation theory correctly predicted formation of large volumes of cartilage within the osteotomy gap and many, though not all patterns of tissue formation observed throughout the callus. The results support the concept that interstitial fluid velocity and tissue shear strain are key mechanical stimuli for the differentiation of skeletal tissues.

Fracture-healing is regulated in part by mechanical factors. Study of the processes by which the mechanical environment of a fracture modulates healing can yield new strategies for the treatment of bone injuries. This article focuses on several key unanswered questions in the study of mechanotransduction and fracture repair. These questions concern identifying the mechanical stimuli that promote bone-healing, defining the mechanisms that are involved in this process, and examining the potential for cross-talk between investigations of mechanotransduction in bone-healing and in healing of other mesenchymally derived tissues. Several approaches to obtain accurate estimates of the mechanical stimuli present within a fracture callus are proposed, and our current understanding of the mechanotransduction processes involved in bone-healing is reviewed. Further study of mechanotransduction mechanisms is needed in order to identify those that are most critical and active during the various phases of fracture repair. A better understanding of the effect of mechanical factors on bone-healing will also benefit the study of healing, regeneration, and engineering of other skeletal tissues. The Mechanical Environment of a Healing FractureFracture-healing is governed by genetic as well as epigenetic factors. The mechanical environment of a healing fracture is one such epigenetic factor that is known to have a profound influence on the rate and success of the repair process. Understanding the effect of the mechanical environment, and in particular the mechanisms by which mechanical cues modulate bone-healing, has applications ranging from clinical management of fractures to bone-tissue engineering and basic science investigations of cell fate.Multiple parameters contribute to the mechanical environment of a fracture callus. These include the stability of fixation, the geometry or type of fracture, and the type of loading. For example, highly stable fixation, such as that provided by a rigidly applied internal fixation plate and by an interfragmentary screw, results in primary cortical healing without the formation of a callus. Less stable external fixation results in a cartilaginous callus, the size of which depends heavily on the stiffness of the fixator frame 1-3 . The geometry or type of fracture affects how the external loads are transferred to the callus tissue. A simple example is the comparison of a transverse fracture line to an oblique fracture line. Even under the same axial compressive Corresponding author: Elise F. Morgan, PhD, Department of Aerospace and Mechanical Engineering, Boston University, 110 Cummington Street, Boston, MA 02215. E-mail address: efmorgan@bu.edu. Disclosure: In support of their research for or preparation of this work, one or more of the authors received, in any one year, outside funding or grants in excess of $10,000 from the National Institutes of Health (grant #AR053353) and the Whitaker Foundation (graduate fellowship) and of less than $10,000 from Boston University (undergraduate ...

Formation of a cartilaginous soft callus at the site of a bone fracture is a pivotal stage in the healing process. Noninvasive, or even nondestructive, imaging of soft callus formation can be an important tool in experimental and pre-clinical studies of fracture repair. However, the low X-ray attenuation of cartilage renders the soft callus nearly invisible in radiographs. This study utilized a recently developed, cationic, iodinated contrast agent in conjunction with micro-computed tomography to identify cartilage in fracture calluses in the femora of C57BL/6J and C3H/HeJ mice. Fracture calluses were scanned beforeand after incubation in the contrast agent. The set of pre-incubation images was registered against and then subtracted from the set of post-incubation images, resulting in a three-dimensional map of the locations of cartilage in the callus, as labeled by the contrast agent. This map was then compared to histology from a previous study. The results showed that the locations where the contrast agent collected in relatively high concentrationswere similar to those of the cartilage. The contrast agent also identified a significant difference between the two strains of mice in the percentage of the callus occupied by cartilage, indicating that this method of contrast-enhanced computed tomography may be an effective technique for nondestructive, early evaluation of fracture healing.

Assessment of the early stages of fracture healing via X-rays and computed tomography is limited by the low radio-opacity of cartilage. We validated a method of contrast-enhanced computed tomography (CECT) for non-destructive identification of cartilage within a healing fracture callus. Closed, stabilized fractures in femora of C57BL/6 mice were harvested on post-operative day 9.5 and imaged ex vivo with micro-computed tomography (μCT) before and after incubation in a cationic contrast agent that preferentially accumulates in cartilage due to the high concentration of sulfated glycosaminoglycans in the tissue. Co-registration of the pre- and post-incubation images, followed by image subtraction, enabled two- and three-dimensional delineation of mineralized tissue, soft callus, and cartilage. The areas of cartilage and callus identified with CECT were compared to those identified with the gold-standard method of histomorphometry. No difference was found between the areas of cartilage measured by the two methods (p = 0.999). Callus area measured by CECT was smaller than, but strongly predictive of (R2 = 0.80, p < 0.001), the corresponding histomorphometric measurements. CECT also enabled qualitative identification of mineralized cartilage. These findings indicate that the CECT method provides accurate, quantitative, and non-destructive visualization of the shape and composition of the fracture callus, even during the early stages of repair when little mineralized tissue is present. The non-destructive nature of this method would allow subsequent analyses, such as mechanical testing, to be performed on the callus, thus enabling higher-throughput, comprehensive investigations of bone healing.

Nearly 10% of the approximately six million fractures that occur each year in the United States do not heal, causing lasting pain and repetitive injury [1]. Although the causes of poor healing are unknown in many cases, the sensitivity of the repair process to mechanical factors is well established. In an effort to understand how mechanical factors such as axial and shear micromotion at the fracture site affect healing, prior studies have sought to characterize the local mechanical environment using finite element (FE) analysis (e.g., [2,3]). However, a key set of inputs for the FE analyses is the distribution of material properties of the various tissues that comprise the fracture callus. Recent studies using nano- and microindentation have estimated these properties by approximating the tissues as linear elastic [4,5]. As a next step in this line of inquiry, the overall goal of this study was to estimate the linear, poroelastic material properties of callus tissues. The specific objectives were: 1) to develop an FE model for use in simulating microindentation experiments; and 2) to compare the results of the simulation to experimental microindentation data in order to derive the mechanical properties of the healing tissues.