Quantitative Assessment of Type-1 Collagen in Charcot Neuroarthropathy with Use of Harmonic Generation and Multiphoton Microscopy

Abstract

Category: Diabetes Introduction/Purpose: Diabetic Charcot neuroarthropathy is characterized by rapid loss of bone with accompanied disruption to the surrounding joints and is often in the absence of trauma or infection. Previous research has demonstrated neuropeptide release and osteoclast activation in addition to increased collagen turnover. Currently, there is limited understanding on the impact of the disease on type-1 collagen within the joint synovium. The purpose of this study was to analyze qualitative and quantitative differences in type-1 collagen between diabetic, neuropathic patient controls and those with Charcot neuroarthropathy. Methods: A prospective case-control study using routinely discarded surgical waste synovial tissue from diabetic, neuropathic patients with and without Charcot. The tissue was prepared in the laboratory for analysis. Microscopy was performed using multiphoton excitation fluorescence (MPEF) and second harmonic generation (SHG) microscopy techniques, utilizing a mode locked femto-second i: Sapphire Tsunami (Spectra-Physics, Mountain View, CA) synchronously pumped by a Millenia Xs J (Spectra-Physics) diode-pumped solid state laser. Qualitative and quantitative microscopic analysis was performed on Charcot specimens and controls. A statistical analysis was performed to detect a quantitative difference in type-1 collagen. Results: An analysis was performed on 8 samples; 4 neuropathic controls and 4 Charcot neuroarthropathy samples. There was a statistically significant difference between the amount, the quality and organization of type-1 collagen (p<0.0001). The Charcot group had significantly less type-1 collagen when compared to the control (p<0.0001). Qualitative analysis was performed using a mean intensity value. The control group’s mean intensity was 2561.4 nm while the Charcot group’s was 884.1 nm. The Charcot group had a significantly smaller mean intensity indicating poorer quality of collagen (p<0.0001). Orientation index was used to assess the organization of type-1 collagen in the synovium. A smaller orientation index signifies an increase in disorganization. The Charcot group had a significantly smaller orientation index of 16.5 compared to the control group’s 27.8 (p<0.0001). Conclusion: Charcot neuroarthropathy leads to significant abnormalities in type-1 collagen within the joint synovium when compared to the diabetic controls. Further research will determine if these differences are attributable to neuropeptide release and osteoclastic activation.