Clayton Bratton

Anulus fibrosus tension inhibits degenerative structural changes in lamellar collagen

Mechanical stress is one of the risk factors believed to influence intervertebral disc degeneration. Animal models have shown that certain regimes of compressive loading can induce a cascade of biological effects that ultimately results in cellular and structural changes in the disc. It has been proposed that both cell-mediated breakdown of collagen and the compromised stability of collagen with loss of anular tension could result in degradation of lamellae in the anulus fibrosus (AF). To determine whether this may be important in the AF, we subjected entire rings of de-cellularized AF tissue to MMP-1 digestion with or without tension. Biomechanical testing found trends of decreasing strength and stiffness when tissues were digested without tension compared with those with tension. To determine the physiologic significance of tissue level tension in the AF, we used an established in vivo murine model to apply a disc compression insult known to cause degeneration. Afterward, that motion segment was placed in fixed-angle bending to impose tissue level tension on part of the AF and compression on the contralateral side. We found that the AF on the convex side of bending retained a healthy lamellar appearance, while the AF on the concave side resembled tissues that had undergone degeneration by loading alone. Varying the time of onset and duration of bending revealed that even a brief duration applied immediately after cessation of compression was beneficial to AF structure on the convex side of bending. Our results suggest that both cell-mediated events and cell-independent mechanisms may contribute to the protective effect of tissue level tension in the AF.

Quantitative analysis of structural disorder in intervertebral disks using second harmonic generation imaging: comparison with morphometric analysis

A novel signal processing algorithm for quantifying structural disorder in biological tissue using second harmonic generation (SHG) imaging is described. Both the magnitude and the pattern of disorder in collagenous tissues can be determined with this method. Mathematical models are used to determine the range of disordered states over which the algorithm can be used, because highly disordered biological samples do not generate second harmonic signals. The method is validated by measuring disorder in heated fascicles using SHG and showing that results are significantly correlated with morphometric determination. Applicability of the method to tissue pathology is demonstrated by analysis of a mouse model of intervertebral disk injury. Disks were subjected to tensile or compressive forces in vivo for one week. Structural disorder in the annulus fibrosus was measured by SHG scanning and by standard morphometric analysis. Values for disorder obtained by SHG scanning were significantly correlated with values obtained by morphometry (p<0.001). Quantitation of disorder using SHG offers significant advantages over morphometric determination. Data obtained in this study suggest that this method can be used to discriminate between reversible and irreversible tissue damage.

Polarization-modulated second harmonic generation imaging: method for quantitative assessment of disorganization in anulus

An experimental method for quantifying disorder within the anulus fibrosus is described based on polarization-modulated second harmonic generation imaging (PM-SHG-I). This method is demonstrated by imaging the anular lamellar architecture of a mouse model of compressive loading. Results were consistent with those obtained in an earlier study where organization was quantified directed secants image analysis on photomicrographs. In this study the orientation within individual lamellia is quantified by average orientation of the collagen molecules within a defined volume of a single lamellar as measured by the PM-SHG-I. Lamellar boundaries can be identified through the SHG intensity images, and confirmed through co-registration with photomicrographs of the same region. The orientation within the lamellar is quantified by the polarization angle of the maximum second harmonic intensity. PM-SHG-I offers several advantages as compared with the method of directed secants: first, it is nondestructive, allowing repeated measurements of the same tissue; second, images are captured on the order of seconds and capable of obtaining information up to a depth of 200-300 microns, thus allowing for real-time assessment of load damage; third, organization is measured at a much higher resolution, as it is based on disorder within the molecular arrays of a single lamella.

First ReFerence photosensor prototype

First test results of the ReFerence photosensor prototype are presented. The results verify the basic design feature, i.e. the photoelectron focusing from the entire photocathode to the small region in the middle of the entrance window of the ReFerence tube.

Measurement of order-disorder transitions in biological samples using polarization-modulated second harmonic generation imaging

Transitions in order/disorder phase states of biological tissues were analyzed using polarization modulated second harmonic generation imaging. The degree of structural disorder was significantly correlated with severity of pathological changes.

Achiral and chiral sum frequency vibrational spectroscopy of collagen fibrils

The noncentrosymmetric molecular structures responsible for the second order nonlinear effects of collagen fibrils are identified by sum frequency vibrational spectroscopy. CH2 and C=O functional groups dominate the achiral and chiral spectra, respectively.