Further understanding of how mechanical cues modulate skeletal tissue differentiation can identify potential means of enhancing repair following injury or disease. Prior studies examined the effects of mechanical loading on osteogenesis, chondrogenesis, and fibrogenesis in an effort to enhance bony union. However, exploring how mechanical stimuli can divert the bone healing process towards formation of other mesenchymal tissues, as an endpoint, may elucidate new avenues for repair and regeneration of tissues such as cartilage and fibrous tissue. This study investigated the use of mechanical stimulation to promote cartilage rather than bone formation within an osteotomy. Our overall goal was to define skeletal tissue distribution and molecular expression patterns induced by the stimulation. Retired breeder Sprague-Dawley rats (n ¼ 85) underwent production of a mid-diaphyseal, transverse femoral osteotomy followed by external fixation. Beginning on postoperative day 10 and continuing for 1, 2, or 4 weeks, a cyclic bending motion (þ358/À258 at 1 Hz) was applied in the sagittal plane for 15 min/day for 5 consecutive days/week. Control animals experienced continuous rigid fixation. Histological and molecular analyses indicated that stimulation substantially altered normal bone healing. Stimulated specimens exhibited an increase in cartilage volume over time, while control specimens demonstrated bony bridging. Stimulation induced upregulation of cartilage-related genes (COL2A1 and COL10A1) and downregulation of bone morphogenetic proteins (BMPs) -4, -6 and -7. However, BMP-3 was upregulated with stimulation. These findings illustrate that mechanical cues can selectively modulate osteogenesis and chondrogenesis in vivo, and suggest a potential basis for treatment regimens for injured or diseased cartilaginous tissues. ß

Defining how mechanical cues regulate tissue differentiation during skeletal healing can benefit treatment of orthopaedic injuries and may also provide insight into the influence of the mechanical environment on skeletal development. Different global (i.e., organ-level) mechanical loads applied to bone fractures or osteotomies are known to result in different healing outcomes. However, the local stimuli that promote formation of different skeletal tissues have yet to be established. Finite element analyses can estimate local stresses and strains but require many assumptions regarding tissue material properties and boundary conditions. This study used an experimental approach to investigate relationships between the strains experienced by tissues in a mechanically stimulated osteotomy gap and the patterns of tissue differentiation that occur during healing. Strains induced by the applied, global mechanical loads were quantified on the mid-sagittal plane of the callus using digital image correlation. Strain fields were then compared to the distribution of tissue phenotypes, as quantified by histomorphometry, using logistic regression. Significant and consistent associations were found between the strains experienced by a region of the callus and the tissue type present in that region. Specifically, the probability of encountering cartilage increased, and that of encountering woven bone decreased, with increasing octahedral shear strain and, to a lesser extent, maximum principal strain. Volumetric strain was the least consistent predictor of tissue type, although towards the end of the four-week stimulation timecourse, cartilage was associated with increasingly negative volumetric strains. These results indicate that shear strain may be an important regulator of tissue fate during skeletal healing.

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 ...

Skeletal repair and regeneration involve a dynamic interplay of biological processes that result in spatially and temporally varying patterns of tissue formation and remodeling. For example, during bone fracture healing the cartilaginous callus that is formed initially in the fracture site is subsequently mineralized and remodeled to restore the original form and function to the injured bone. During much of this healing process, the fracture callus is comprised of a heterogeneous mixture of cartilage, fibrocartilage, multipotent mesenchymal tissue, and bone. Adding to this complexity, mechanical stimuli are known to influence the rate and type of tissues formed during skeletal healing [1]. Given the growing body of evidence that controlled mechanical stimulation may be used to enhance healing, it is of substantial interest to elucidate relationships between the distributions of local stresses and strains that develop within the healing region and the distribution of tissue types that form. While histomorphometry is a well established approach for characterizing the latter, it has historically been limited to analyses of a small number of two-dimensional sections of tissue. Such 2D sampling may be inadequate for quantitative characterization of the irregular geometry and heterogeneous composition of healing tissues. In this study, we report on a 3D histomorphometric method and apply this method to an in vivo model of skeletal repair [2] in which a bending stimulus delivered to a healing bone defect results in the formation of predominantly cartilage tissue, rather than bone.