Tissue engineers rely on expensive, time-consuming, and destructive techniques to monitor the composition 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. A label-free multimodal system combining fluorescence lifetime imaging (FLIm) and optical coherence tomography (OCT) via a single fiber-optic interface was used for evaluation of biochemical and structural properties of tissue-engineered articular cartilage in a murine model of cartilage maturation. Nude mice (n=5) received 2 dorsal subcutaneous tissue-engineered cartilage implants each consisting of: 1) latent transforming growth factor-beta1 (LAP) treated; and 2) untreated control (CTL) constructs. At 6 weeks post-implantation, mice were sacrificed and multimodal imaging was performed in situ. FLIm showed clear delineation of the implant in all spectral bands (SB). Quantification of the cartilage construct fluorescence lifetime (LT) showed a lower LT in SB-1 (375-410 nm) and higher SB-3 LT (515-565 nm) as compared to the surrounding muscle tissue. Comparison between treatment groups showed a significant increase in FLIm SB-3 LT in LAP-treated constructs over CTL (p < 0.01). Quantification of OCT images allowed implant morphology and 3D volume comparisons between treatment groups. These results suggest that FLIm-OCT based tools are a potential non-destructive method for quantitatively monitoring the growth and quality of tissue engineered articular cartilage. The use of optical techniques to monitor maturation could represent a significant element in reducing costs in research, meeting the FDA regulatory requirements for manufacturing, and providing novel diagnostic tools in the clinic.
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.
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.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.