Pilot study for detection of early changes in tissue associated with heterotopic ossification: moving toward clinical use of Raman spectroscopy

<div><p></p><p>Over 60% of combat-wounded patients develop heterotopic ossification (HO). Nearly 33% of them require surgical excision for symptomatic lesions, a procedure that is both fraught with complications and can delay or regress functional rehabilitation. Relative medical contraindications limit widespread use of conventional means of primary prophylaxis, such as nonspecific nonsteroidal anti-inflammatory medications and radiotherapy. Better methods for risk stratification are needed to both mitigate the risk of current means of primary prophylaxis as well as to evaluate novel preventive strategies currently in development. We asked whether Raman spectral changes, measured <i>ex vivo</i>, could be associated with histologic evidence of the earliest signs of HO formation and substance P (SP) expression in tissue biopsies from the wounds of combat casualties. In this pilot study, we compared normal muscle tissue, injured muscle tissue, very early HO lesions ( < 16 d post-injury), early HO lesions ( > 16 d post-injury) and mature HO lesions. The Raman spectra of these tissues demonstrate clear differences in the Amide I and III spectral regions of HO lesions compared to normal tissue, denoted by changes in the Amide I band center (<i>p</i> < 0.01) and the 1340/1270 cm⁻¹ (<i>p</i> < 0.05) band area and band height ratios. SP expression in the HO lesions appears to peak between 16 and 30 d post-injury, as determined by SP immunohistochemistry of corresponding tissue sections, potentially indicating optimal timing for administration of therapeutics. Raman spectroscopy may therefore prove a useful, non-invasive and early diagnostic modality to detect HO formation before it becomes evident either clinically or radiographically.</p></div

Functional outcomes in sham and experimental ischemia groups.

<p>A) Mean drop foot scores for all experimental groups. A drop foot score of 0 meant that no neuropathy was observed post-operatively. A drop foot score of 1 was consistent with neuropathy in the affected limb for at least one day post-operatively. A drop foot score of 2 was consistent with neuropathy in the affected limb for at least three days post-operatively. A drop foot score of 3 was indicative of neuropathy in the affected limb beyond 5 days post-operatively. B) Mean Tarlov scores for all experimental groups. The dashed line at 5.0 is reflective of normal locomotion. The asterisks indicate statistically significant differences when compared to sham and 3.5 hour occlusion animals—** = p-value < 0.01, *** = p-value < 0.001.</p

Slopes of reperfusion and post-occlusive reactive hyperemia for 3CCD and IR imaging.

<p>Bar plots comparing the IR slopes of reperfusion for all experimental groups (A) and 3CCD slopes of reperfusion, slopes of PORH at 5 minutes reperfusion, and slopes of PORH at 10 minutes reperfusion for all experimental groups (B). Statistically significant differences between 4.7 hour tourniquet animals and other experimental groups are designated by an asterisk (*)–p-value<0.05. Note, for each set of slopes presented in (B), the data is plotted by lowest Tarlov score (i.e. least normal locomotion) and increases to the highest Tarlov score (i.e. most normal locomotion). 3_5O = 3.5 hour occlusion; 3_5T = 3.5 hour tourniquet; 4_7O = 4.7 hour occlusion; 4_7T = 4.7 hour tourniquet.</p

Profiles of operative 3CCD and IR imaging values.

<p>Bar plots of mean R-B values derived from 3CCD imaging (A) and mean leg temperatures in °F (B) for all experimental groups at baseline, maximum ischemia, 10 minutes post-reperfusion, and 30 minutes post-reperfusion. Error bars = SEM (standard error of the mean). 3_5O = 3.5 hour occlusion; 3_5T = 3.5 hour tourniquet; 4_7O = 4.7 hour occlusion; 4_7T = 4.7 hour tourniquet.</p

3CCD and infrared (IR) imaging. Top) Representative grayscale 3CCD images of a hind limb, and Middle) representative grayscale infrared images of a hind limb at (1) baseline, (2) maximum ischemia, (3) 10 minutes post-reperfusion, and (4) 30 minutes post-reperfusion.

<p>Bottom left) Profile of R-B values, derived from 3CCD imaging, over the course of 3.5 hours of ischemia and 30 minutes of reperfusion. Bottom right) Profile of IR values and corresponding mean leg temperature over the course of 3.5 hours of ischemia and 30 minutes of reperfusion. The time points mentioned previously (1–4) are noted.</p

Mean pathology scores for all experimental groups.

<p>Mean total pathology scores for all experimental groups: A) organs (kidneys and liver), and B) sciatic nerve and skeletal muscle. 3_5O = 3.5 hour occlusion; 3_5T = 3.5 hour tourniquet; 4_7O = 4.7 hour occlusion; 4_7T = 4.7 hour tourniquet.</p

Correlation of imaging parameters with blood chemistries.

<p>Pearson's correlation coefficients (ρ) for parameters involving blood chemistries. IR = infrared imaging; Rpf = Rpfusion; 3CCD = 3CCD imaging; PORH = post-occlusive reactive hyperemia; D = day.</p

Outcome based comparison of various metrics.

<p>Values are means +/- the standard deviation for animals that 1) recovered full locomotion, and 2) did not recover full locomotion. Differences approaching statistical significance (p-value ≈ 0.1) are indicated by (†). Statistically significant differences (p-value<0.05) are indicated by an asterisk (*).</p

PLSDA models to predict return to normal locomotion for all data, imaging only data, and non-imaging data sets.

<p>Receiver operating curves for both calibration and cross-validation data sets. Area under the curve (AUC), a surrogate for accuracy, is displayed for each curve. A) PLSDA model generated from all parameters, but excluding outcome parameters. B) PLSDA model calculated from only imaging parameters. C) PLSDA model generated from all non-imaging parameters, but excluding outcome parameters. The calibration data is plotted in black and the cross-validation data is plotted in gray.</p

Characterization of Cells Isolated from Genetic and Trauma-Induced Heterotopic Ossification

<div><p>Heterotopic ossification (HO) is the pathologic formation of bone separate from the normal skeleton. Although several models exist for studying HO, an understanding of the common <i>in vitro</i> properties of cells isolated from these models is lacking. We studied three separate animal models of HO including two models of trauma-induced HO and one model of genetic HO, and human HO specimens, to characterize the properties of cells derived from tissue containing pre-and mature ectopic bone in relation to analogous mesenchymal cell populations or osteoblasts obtained from normal muscle tissue. We found that when cultured <i>in vitro</i>, cells isolated from the trauma sites in two distinct models exhibited increased osteogenic differentiation when compared to cells isolated from uninjured controls. Furthermore, osteoblasts isolated from heterotopic bone in a genetic model of HO also exhibited increased osteogenic differentiation when compared with normal osteoblasts. Finally, osteoblasts derived from mature heterotopic bone obtained from human patients exhibited increased osteogenic differentiation when compared with normal bone from the same patients. These findings demonstrate that across models, cells derived from tissues forming heterotopic ossification exhibit increased osteogenic differentiation when compared with either normal tissues or osteoblasts. These cell types can be used in the future for <i>in vitro</i> investigations for drug screening purposes.</p></div