Nonlinear Optical Microscopy Assessment of Tissue Structure and Chondrocyte Viability of Articular Cartilage

Author

Michael LeFollow

Date of Award

5-2022

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Bioengineering

Committee Chair/Advisor

Tong Ye

Committee Member

Ann Foley

Committee Member

Yongren Wu

Abstract

Articular cartilage functions to protect the ends of bones by providing a surface that can withstand compressive forces and minimize friction during movement. Collagen fibers form the organizational backbone of the extracellular matrix (ECM) in cartilage. Proteoglycans within the ECM function to retain water and provide the tissue with the swelling pressure needed to withstand compressional forces. Chondrocytes, the only type of cell found in articular cartilage, produces these collagen fibers and proteoglycans to maintain the tissue structure and function. Significant injuries to articular cartilage can damage the chondrocytes and disrupt their ability to maintain homeostasis in the tissue. Therefore, chondrocyte viability is an important factor in assessing the severity of cartilage injury or the progression of degenerative joint diseases.

Assessing chondrocyte viability typically involves dye-labeling their cellular and ECM structures. Unfortunately, these methods disrupt the metabolism of the chondrocytes and cannot be used in patients or for longitudinal studies. Using nonlinear optical imaging methods, specifically two-photon excitation fluorescence (TPEF) and second-harmonic generation (SHG), chondrocyte viability and ECM structures can be nondestructively evaluated through the measurement of intrinsic signals from endogenous fluorophores. We have demonstrated a method that uses endogenous fluorophores as intrinsic biomarkers to assess chondrocyte viability and collagen organization without the need for histological dyes.

This thesis aims to validate our chondrocyte viability assay and extend this technology to study the structural and function changes in damaged cartilage. First, we imaged articular cartilage from porcine femoral and tibial condyles using TPEF and SHG microscopy and analyzed the chondrocyte organization and ECM structure. We observed structural differences at different loading regions, implying that these structural differences may correlate to different mechanical properties at different locations. We then performed imaging experiments to validate our autofluorescence-based viability assay in porcine cartilage. Sensitivity and specificity analysis of our results against a calcein AM and ethidium homodimer dye-labeling viability assay confirmed the reliability and applicability of our assay to porcine cartilage. Finally, we developed longitudinal imaging methods to dynamically assess changes to tissue structure and chondrocyte viability in articular cartilage after mechanical loading. We designed a custom tissue culture/imaging chamber to follow the structural changes in the tissue up to several weeks. We also built a mechanical loading instrument that fits inside a biosafety hood to avoid tissue contamination while applying mechanical loading cycles. Preliminary experiments showed success in minimizing contamination during culturing, mechanical loading, and imaging procedures.

Recommended Citation

Le, Michael, "Nonlinear Optical Microscopy Assessment of Tissue Structure and Chondrocyte Viability of Articular Cartilage" (2022). All Theses. 3717.
https://open.clemson.edu/all_theses/3717