James Macione

Collagen Fibrils in Skin Orient in the Direction of Applied Uniaxial Load in Proportion to Stress while Exhibiting Differential Strains around Hair Follicles

We determined inhomogeneity of strains around discontinuities as well as changes in orientation of collagen fibrils under applied load in skin. Second Harmonic Generation (SHG) images of collagen fibrils were obtained at different strain magnitudes. Changes in collagen orientation were analyzed using Fast Fourier Transforms (FFT) while strain inhomogeneity was determined at different distances from hair follicles using Digital Image Correlation (DIC). A parameter, defined as the Collagen Orientation Index (COI), is introduced that accounts for the increasingly ellipsoidal nature of the FFT amplitude images upon loading. We show that the COI demonstrates two distinct mechanical regimes, one at low strains (0%, 2.5%, 5% strain) in which randomly oriented collagen fibrils align in the direction of applied deformation. In the second regime, beginning at 5% strain, collagen fibrils elongate in response to applied deformation. Furthermore, the COI is also found to be linearly correlated with the applied stress indicating that collagen fibrils orient to take the applied load. DIC results indicated that major principal strains were found to increase with increased load at all locations. In contrast, minimum principal strain was dependent on distance from hair follicles. These findings are significant because global and local changes in collagen deformations are expected to be changed by disease, and could affect stem cell populations surrounding hair follicles, including mesenchymal stem cells within the outer root sheath.

Design and analysis of a novel mechanical loading machine for dynamic in vivo axial loading.

This paper describes the construction of a loading machine for performing in vivo, dynamic mechanical loading of the rodent forearm. The loading machine utilizes a unique type of electromagnetic actuator with no mechanically resistive components (servotube), allowing highly accurate loads to be created. A regression analysis of the force created by the actuator with respect to the input voltage demonstrates high linear correlation (R(2) = 1). When the linear correlation is used to create dynamic loading waveforms in the frequency (0.5-10 Hz) and load (1-50 N) range used for in vivo loading, less than 1% normalized root mean square error (NRMSE) is computed. Larger NRMSE is found at increased frequencies, with 5%-8% occurring at 40 Hz, and reasons are discussed. Amplifiers (strain gauge, linear voltage displacement transducer (LVDT), and load cell) are constructed, calibrated, and integrated, to allow well-resolved dynamic measurements to be recorded at each program cycle. Each of the amplifiers uses an active filter with cutoff frequency at the maximum in vivo loading frequencies (50 Hz) so that electronic noise generated by the servo drive and actuator are reduced. The LVDT and load cell amplifiers allow evaluation of stress-strain relationships to determine if in vivo bone damage is occurring. The strain gauge amplifier allows dynamic force to strain calibrations to occur for animals of different sex, age, and strain. Unique features are integrated into the loading system, including a weightless mode, which allows the limbs of anesthetized animals to be quickly positioned and removed. Although the device is constructed for in vivo axial bone loading, it can be used within constraints, as a general measurement instrument in a laboratory setting.

Measurement of lacunar bone strains and crack formation during tensile loading by digital volume correlation of second harmonic generation images

The maintenance of healthy bone tissue depends upon the ability of osteocytes to respond to mechanical cues on the cellular level. The combination of digital volume correlation and second harmonic generation microscopy offers the opportunity to investigate the mechanical microenvironment of intact bone on the scale of individual osteocytes. Adult human femurs were imaged under tensile loads of 5 and 15MPa and volumes of approximately 492×429×31μm(3) were analyzed, along with an image of a bone microcrack under the same loading conditions. Principal strains were significantly higher in three-dimensional digital volume correlation when compared to two-dimensional digital image correlation. The average maximum principal strain magnitude was 5.06-fold greater than the applied global strain, with peak strains of up to 23.14-fold over global strains measured at the borders of osteocyte lacunae. Finally, a microcrack that initiated at an osteocyte lacunae had its greatest tensile strain magnitudes at the crack expansion front in the direction of a second lacunae, but strain at the crack border was reduced to background strain magnitudes upon breaching the second lacunae. This serveed to demonstrate the role of lacunae in initiating, mediating and terminating microcrack growth.

Bone strain measurement using 3D digital image correlation of second harmonic generation images

Collagen fibrils contribute to the structural integrity and crack resistance of bone, but their response to stress while in bundles of fibrils is not well known. Digital image correlation (DIC) is a powerful technique for measuring strain by comparing images of deformed samples to non-deformed samples, while second harmonic generation microscopy (SHGM) captures bright, high contrast images of bone with strong signals from collagen fibrils. Combining both techniques allows the 3-dimensional strain environments of bundled collagen fibrils to be characterized. SHGM was performed using confocal microscopy, producing images of human femur bone at 5 MPa and 50 MPa of applied tensional stress with 0.5×0.5×1 µm voxels. A DIC algorithm generated preliminary displacement and strain maps from the images, with the average calculated strain varying from the theoretical strain. The local strain average in the direction of applied stress was 1.09%, compared to a global strain of 0.627%, which was attributed to the small volume of analysis compared to the global image, and the proximity of a lacuna to the analysis region. The strain response was also found to be highly heterogeneous. Future work will analyze larger regions of bone.

Magnitude of loads influences the site of failure of highly curved bones

The structure and material properties of bones along with applied boundary conditions determine the region of peak stresses, where fracture is expected to occur. As the site of peak stresses is not influenced by the magnitude of applied load, the fracture site is not expected to change during fatigue loading of whole bone at different loads. However, in a highly curved bone such as the rat ulna, the magnitude of applied loads was found to influence the fracture site. Fatigue loading was conducted under load control on intact rat forearms and on excised ulnae. The distance to the site of failure from the proximal olecranon process of ulnae was determined. In intact forearms, the site of failure demonstrated a linear progression distally, towards sites with lower moment of inertia (or sites exhibiting lower section modulus). Intact rat forearms and excised ulnae loaded to failure at low loads fractured 2-3mm distal to where they failed when applying high loads. This indicates a shift in the site of failure by approximately 10% of whole bone length just by varying the applied load magnitude. The site of failure in excised ulnae was similar when loading at 2Hz or at 4Hz, suggesting that this was frequency independent in this range and indicating that strain rate was not an important contributing factor. Creep loading of excised ulnae also demonstrated similar changes in the site of failure, indicating that magnitude of loads, and not type of loading were important in determining the site of failure. This has important implications with regards to the volume of bone that undergoes damage under physiological loading, before it fails.

Reprogramming of cells using modified mRNA

Reprogramming of cells for delivery of proteins at specific sites holds great promise for future clinical applications. Chitosan delivery agents in form of nano-spheres presents a readily available biodegradable option for controlled release of mRNA in the cytosol of the cells. In this work we are using Enhanced Green Fluorescent protein as a proof of concept to show for transfection of osteoblast cells with modified mRNA. Transfection was carried out using lipofectaime RNAiMAX.

Mapping of Strains In Bundles Of Skin Collagen Microfibrils

Collagen is believed to be one of the principal load bearing components of skin. This implies that deformation of collagen microfibrils play an important role in determining the extent of skin deformation under applied loads. We have developed a novel technique for generating internal strain maps of bundles of collagen microfibrils in the skin. The technique relies on digital image correlation (DIC) of second harmonic images of bundles of collagen fibrils obtained under different deformations. Applying the technique to mouse skin demonstrated a significant heterogeneity of deformation in the underlying collagen microfibrils. This technique can be used to evaluate the contribution of collagen to the biomechanical properties of skin in aging, disease, as well as in wound healing.