Non-destructive, continuous monitoring of biochemical, mechanical, and structural maturation in engineered tissue.

Regulatory guidelines for tissue engineered products require stringent characterization during production and necessitate the development of novel, non-destructive methods to quantify key functional parameters for clinical translation. Traditional assessments of engineered tissues are destructive, expensive, and time consuming. Here, we introduce a non-destructive, inexpensive, and rapid sampling and analysis system that can continuously monitor the mechanical, biochemical, and structural properties of a single sample over extended periods of time. The label-free system combines the imaging modalities of fluorescent lifetime imaging and ultrasound backscatter microscopy through a fiber-based interface for sterile monitoring of tissue quality. We tested the multimodal system using tissue engineered articular cartilage as an experimental model. We identified strong correlations between optical and destructive testing. Combining FLIm and UBM results, we created a novel statistical model of tissue homogeneity that can be applied to tissue engineered constructs prior to implantation. Continuous monitoring of engineered tissues with this non-destructive system has the potential for in-process monitoring of tissue engineered products, reducing costs and improving quality controls in research, manufacturing, and clinical applications.

Multiscale, multispectral fluorescence lifetime imaging using a double-clad fiber.

Fiber-based imaging of tissue autofluorescence using ultraviolet (UV) excitation is a highly flexible tool used to probe structure and composition. In this Letter, we report, to the best of our knowledge, the first results from a single-fiber imaging system employing a custom double-clad fiber to acquire multispectral fluorescence lifetime images at two distinct spatial resolutions. We characterize the lateral point spread function and fluorescent background of the system and show how enhanced resolution can identify features such as trabeculae in ex vivo murine bone samples.

Non-destructive detection of matrix stabilization correlates with enhanced mechanical properties of self-assembled articular cartilage.

Tissue engineers rely on expensive, time-consuming, and destructive techniques to monitor the composition, microstructure, and function of engineered tissue equivalents. A non-destructive solution to monitor tissue quality and maturation would greatly reduce costs and accelerate the development of tissue-engineered products. The objectives of this study were to (a) determine whether matrix stabilization with exogenous lysyl oxidase-like protein-2 (LOXL2) with recombinant hyaluronan and proteoglycan link protein-1 (LINK) would result in increased compressive and tensile properties in self-assembled articular cartilage constructs, (b) evaluate whether label-free, non-destructive fluorescence lifetime imaging (FLIm) could be used to infer changes in both biochemical composition and biomechanical properties, (c) form quantitative relationships between destructive and non-destructive measurements to determine whether the strength of these correlations is sufficient to replace destructive testing methods, and (d) determine whether support vector machine (SVM) learning can predict LOXL2-induced collagen crosslinking. The combination of exogenous LOXL2 and LINK proteins created a synergistic 4.9-fold increase in collagen crosslinking density and an 8.3-fold increase in tensile strength as compared with control (CTL). Compressive relaxation modulus was increased 5.9-fold with addition of LOXL2 and 3.4-fold with combined treatments over CTL. FLIm parameters had strong and significant correlations with tensile properties (R2  = 0.82; p < 0.001) and compressive properties (R2  = 0.59; p < 0.001). SVM learning based on FLIm-derived parameters was capable of automating tissue maturation assessment with a discriminant ability of 98.4%. These results showed marked improvements in mechanical properties with matrix stabilization and suggest that FLIm-based tools have great potential for the non-destructive assessment of tissue-engineered cartilage.

Detection of glycosaminoglycan loss in articular cartilage by fluorescence lifetime imaging.

Glycosaminoglycan (GAG) loss is an early marker of osteoarthritis, which is a clinical late stage disease that affects millions of people worldwide. The goal of our study was to evaluate the ability of a fiber-based fluorescence lifetime imaging (FLIm) technique to detect GAG loss in articular cartilage. Native bovine cartilage explants (n = 20) were exposed to 0 (control), 0.5 (low), or 1 U / mL (high) concentrations of chondroitinase ABC (cABC) to create samples with different levels of GAG loss. FLIm assessment (excitation: 355 nm; detection: channel 1: 375 to 410 nm, channel 2: 450 to 485 nm, channel 3: 530 to 565 nm) was conducted on depth-resolved cross-sections of the cartilage sample. FLIm images, validated with histology, revealed that loss of GAG resulted in a decrease of fluorescence lifetime values in channel 2 (Δ = 0.44 ns, p < 0.05) and channel 3 (Δ = 0.75 ns, p < 0.01) compared to control samples (channel 2: 6.34 ns; channel 3: 5.22 ns). Fluorescence intensity ratio values were lower in channel 1 (37%, p < 0.0001) and channel 2 (31% decrease, p < 0.0001) and higher in channel 3 (23%, p < 0.0001) relative to control samples. These results show that FLIm can detect the loss of GAG in articular cartilage and support further investigation into the feasibility of in vivo FLIm arthroscopy.

Label-Free Assessment of Collagenase Digestion on Bovine Pericardium Properties by Fluorescence Lifetime Imaging.

The extracellular matrix architecture of bovine pericardium (BP) has distinct biochemical and biomechanical properties that make it a useful biomaterial in the field of regenerative medicine. Collagen represents the dominant structural protein of BP and is therefore intimately associated with the properties of this biomaterial. Enzymatic degradation of collagen molecules is critical for extracellular matrix turnover, remodeling and ultimately tissue regeneration. We present a quantitative, label-free and non-destructive method for monitoring changes in biochemical and biomechanical properties of BP during tissue degradation, based on multi-spectral fluorescence lifetime imaging (ms-FLIm). Strong correlations of fluorescence intensity ratio and average fluorescence lifetime were identified with collagen content, Young's Modulus and Ultimate tensile strength during collagenase degradation, indicating the potential of optically monitoring collagen degradation using ms-FLIm. The obtained results demonstrate the value of ms-FLIm to assess the quality of biomaterials in situ for applications in regenerative medicine.

Fiber-based fluorescence lifetime imaging of recellularization processes on vascular tissue constructs.

New techniques able to monitor the maturation of tissue engineered constructs over time are needed for a more efficient control of developmental parameters. Here, a label-free fluorescence lifetime imaging (FLIm) approach implemented through a single fiber-optic interface is reported for nondestructive in situ assessment of vascular biomaterials. Recellularization processes of antigen removed bovine pericardium scaffolds with endothelial cells and mesenchymal stem cells were evaluated on the serous and the fibrous sides of the scaffolds, 2 distinct extracellular matrix niches, over the course of a 7 day culture period. Results indicated that fluorescence lifetime successfully report cell presence resolved from extracellular matrix fluorescence. The recellularization process was more rapid on the serous side than on the fibrous side for both cell types, and endothelial cells expanded faster than mesenchymal stem cells on antigen-removed bovine pericardium. Fiber-based FLIm has the potential to become a nondestructive tool for the assessment of tissue maturation by allowing in situ imaging of intraluminal vascular biomaterials.

Nondestructive assessment of collagen hydrogel cross-linking using time-resolved autofluorescence imaging.

We investigate the use of a fiber-based, multispectral fluorescence lifetime imaging (FLIm) system to nondestructively monitor changes in mechanical properties of collagen hydrogels caused by controlled application of widely used cross-linking agents, glutaraldehyde (GTA) and ribose. Postcross-linking, fluorescence lifetime images are acquired prior to the hydrogels being processed by rheological or tensile testing to directly probe gel mechanical properties. To preserve the sterility of the ribose-treated gels, FLIm is performed inside a biosafety cabinet (BSC). A pairwise correlation analysis is used to quantify the relationship between mean hydrogel fluorescence lifetimes and the storage or Young's moduli of the gels. In the GTA study, we observe strong and specific correlations between fluorescence lifetime and the storage and Young's moduli. Similar correlations are not observed in the ribose study and we postulate a reason for this. Finally, we demonstrate the ability of FLIm to longitudinally monitor dynamic cross-link formation. The strength of the GTA correlations and deployment of our fiber-based FLIm system inside the aseptic environment of a BSC suggests that this technique may be a valuable tool for the tissue engineering community where longitudinal assessment of tissue construct maturation in vitro is highly desirable.

Simultaneous, label-free, multispectral fluorescence lifetime imaging and optical coherence tomography using a double-clad fiber.

We present a novel fiber-based imaging platform that allows simultaneous fluorescence lifetime imaging (FLIm) and optical coherence tomography (OCT) using a double-clad fiber. This platform acquires co-registered images showing structural and compositional contrast in unlabeled biological samples by scanning the fiber tip across the sample surface. In this Letter, we report a characterization of each modality and show examples of co-registered FLIm and OCT images acquired from a lemon segment and a section of human coronary artery. The close comparison between the combined FLIm and OCT images and a co-registered histology section provides a qualitative validation of the technique and highlights its potential for minimally invasive, multimodal imaging of tissue structure and composition.