1.

Introduction

Articular cartilage is the near frictionless tissue at the end of long bones that allows smooth movement of joints and absorbs shock. Cartilage degenerative diseases such as osteoarthritis (OA) are major causes of disability worldwide¹ and have a substantial contribution to healthcare cost.² This large economic burden will continue to increase as the population ages. Clinical OA is a late-stage condition for which disease-modifying opportunities are limited. However, OA typically develops over decades, offering a long window of time to potentially alter its course. As such, characterization of pre- or early-OA disease states will be critical to support a paradigm shift from palliation of late disease toward prevention.³ Unfortunately, early diagnosis of OA is still a challenging, unmet clinical need that must be addressed.

Cartilage extracellular matrix (ECM) is composed primarily of 15% to 30% glycosaminoglycan (GAG) (per dry weight), 50% to 75% collagen (per dry weight), and 70% to 80% water (per wet weight). The remaining balance of dry weight includes minor protein molecules and chondrocytes.^4,5 The mechanical properties of articular cartilage are determined by the biochemical composition of the main tissue constituents, GAG and collagen, creating a structure–function relationship in which GAG resists compressive loading and collagen resists tensile loading within the tissue.⁶ During the early stages of OA, the changes in cartilage are clinically silent. No visual, functional, or mechanical alterations of articular cartilage appear detectable. Gradual GAG loss is the first observable indication of OA. Left untreated, it will lead to a “death spiral” of increasing matrix degradation and reduced biomechanical properties.⁷ As a result, normal loading increases matrix damage, leading to a positive feedback loop of cartilage damage, pain, and destruction. This deterioration continues until the cartilage completely loses its ability to withstand load and is worn away to expose subchondral bone.⁸ Therefore, the detection of GAG loss is critical for the early diagnosis of OA.

Imaging modalities are an integral part of the diagnosis of OA. Conventional imaging modalities like arthroscopic imaging, standard radiography, ultrasonography, and magnetic resonance imaging (MRI) are widely used in daily clinical diagnosis.^9–11 Despite that, arthroscopy is primarily a qualitative assessment technique that falls short of the laboratory assessment standards of histopathology, biochemical analysis, and biomechanical testing. Both radiography and ultrasound fail to provide critical information regarding the biochemical composition of the cartilage ECM. Conventional MRI allows accurate assessment of both cartilage morphology and biochemical composition (collagen ultrastructure and GAG concentration) but still suffers from low clinical image resolution.¹²

Over the past decade, optical imaging modalities have been studied to evaluate the biochemical makeup and structural organization of cartilage and are being actively explored for utility in cartilage research and OA diagnosis. This includes laser scanning confocal microscopy,¹³ second harmonic generation microscopy, multiphoton microscopy,¹⁴ Fourier transform infrared imaging and spectroscopy,¹⁵ and Raman spectroscopy.¹⁶ Unfortunately, challenges, including the complexity and high cost of instrumentation and low data acquisition speed, hamper their clinical translation. Moreover, optical coherence tomography (OCT) allowing evaluation of cartilage microstructure associated with cartilage health has been used arthroscopically as a translational research tool for early diagnosis of OA.^17,18 Still, OCT lacks the ability to provide biochemical information.

Therefore, there is a need for clinical compatible imaging modalities for in vivo assessment of biochemical composition related to cartilage health. Fiber-based time-resolved fluorescence lifetime imaging (FLIm) has the potential to address this need. FLIm with a lower-case m was used as abbreviation to distinguish our fiber-based technology from fluorescence lifetime microscopy (FLIM). Over the past decade, several groups have reported that FLIm may be used for disease diagnosis and tissue characterization with applications in numerous clinical areas, including oncology, cardiology, and ophthalmology.¹⁹ Multimodal instrumentation combining FLIm and ultrasound has also been used to monitor matrix composition and mechanical properties of engineered cartilage tissue during maturation.^20,21 Recent advantages in FLIm devices using miniature fiber optics probes, along with fast electronics enabling fast data acquisition speed, analysis, and real-time display²² make this technique highly compatible with modern clinical arthroscopes. Hence, the goal of the present study was to investigate the feasibility of using FLIm for early detection of cartilage disease. Specifically, this study focused on (1) evaluation of fluorescence properties of native articular cartilage and their variation across different depth-resolved zones, (2) investigation of whether FLIm can detect the depletion of major cartilage biochemical constituents involved in development of OA, namely GAG, and (3) determining whether FLIm parameters can be used to infer the level of GAG loss in cartilage.

2.

Materials and Methods

2.1.

Cartilage Harvest

Articular cartilage was excised as full thickness cylindrical osteochondral plugs ($n=25$) from a juvenile (2-week-old) bovine femoral head obtained from an abattoir (Research 87, Boylston, MA) within 48 h of death. Cartilage explants were wrapped in gauze and soaked in phosphate-buffered saline (PBS) with protease inhibitors (2-nM phenylmethylsulfonyl fluoride, 10-mM N-ethyl malemide, 2-mM ethylene diamine tetraacetatic acid, and 5-mM benzamidine-HCl) as previously described,²³ underwent three freeze thaw cycles to lyse cells, and stored at $-20°\mathrm{C}$ in protease inhibitor until imaging [Fig. 1(a)].

Fig. 1

(a) Schematic diagram of the study protocol. Harvest and treatment of bovine articular cartilage samples in untreated (control) and cABC groups. Zonal en-face imaging was conducted on samples from different depths of cartilage from control group. Cross-sectional imaging was conducted on both control and cABC treatment groups. (b) Schematic diagram of the FLIm system. FLIm images were acquired by raster scanning of the optical fiber. (c) Spectral channels of the FLIm instrument: channel 1 = 375 to 410 nm, channel 2 = 450 to 485 nm, and channel 3 = 530 to 565 nm. The reference emission spectrum of collagen (red) and GAG (blue)²⁴ was also overlaid.

3.

Results

3.1.

Depth-Dependent Lifetime of Native Articular Cartilage

Cross-sectional FLIm images [Fig. 2(a)] showed that fluorescence LT in channel 1 varied with cartilage depth. The region closest to the cartilage surface had a LT of $5.7\pm 0.12\mathrm{ns}$ while the middle and deep part of cartilage had a longer LT of $6.1\pm 0.08\mathrm{ns}$ ($p<0.0001$). The same trend was also presented in the zonal FLIm images [Fig. 2(a)]. This was further confirmed by the violin plot as the distributions of LT from both cross-sectional and zonal imaging shifted to higher LT values in middle and deep zone [Fig. 2(b)]. This trend was observed for all samples as depicted by the box-plot of LT distribution from all samples ($n=3$), which demonstrated that the LT increases with distance from cartilage surface. This increase was most noticeable within $500\mu \mathrm{m}$ from the surface [Fig. 2(c)]. In addition, changes of fluorescence LT were also associated with morphological heterogeneities. Macrostructures with shorter LT were also presented in both cross-section and en-face FLIm LT images [Fig. 2(a)]. No significant depth-dependent LT changes were observed in other spectral channels.

Fig. 2

Channel 1 (375 to 410 nm) fluorescence LT increases with distance from articular cartilage surface ($n=3$). (a) Representative cross-section and zonal FLIm image of native bovine articular cartilage. $\text{Scalebar}=1\mathrm{mm}$. Dashed line represents estimated location of cut of superficial, middle and deep zone. (b) Violin plot of LT distribution of different zones of articular cartilage acquired through zonal and $x$-section imaging. The LT of superficial zone is significantly lower than that of middle and deep zones (***$p<0.001$). (c) A box plot of average LT versus distance from cartilage surface for all samples ($n=3$). Each box represents all data points from all samples at the corresponding distance from cartilage surface. Five values are highlighted: the extremes, the upper and lower quantiles, and the median. A clear increase of LT with distance from cartilage surface is observed. The increase is most prominent within the first 0.5 mm of cartilage surface.

3.2.

Detection of GAG-Depletion using FLIm

Safranin-O stained histology images showed that compared to untreated (control) cartilage, enzymatic treatment of cABC almost completely depleted GAG content within 1 mm distance from cartilage surface [Fig. 3(a)]. The depletion of GAG resulted in significant decreases in fluorescence LT in both detection channel 2 ($p<0.05$) and channel 3 ($p<0.01$) and significant changes in the intensity ratios in all collection channels ($p<0.0001$) [Fig. 3(b)].

Fig. 3

FLIm detects GAG depletion in articular cartilage. (a) Representative fluorescence cross-sectional intensity ratio images, LT images, and histology images of untreated and treated bovine articular cartilage. ROIs highlight the region of GAG depletion in histology images of cABC treated sample. The corresponding region in FLIm LT and intensity ratio images are highlighted by stars. The GAG depletion region appears in FLIm maps with higher intensity ratio and lower LT in channel 3 as well as lower intensity ratio in channel 1. No GAG depletion is observed for control sample. $\text{Scalebar}=1\mathrm{mm}$ ($n\ge 6$ per group). (b) Box plot of ROI average LT from all samples in control and high cABC group. Significant LT differences are observed in channel 2 (450 to 485 nm) *$p<0.05$ and channel 3 (530 to 565 nm) **$p<0.01$. Significant differences in intensity ratio are observed in all channels. ****$p<0.0001$.

A segmentation algorithm based on channel 1 intensity ratio was able to identify regions of GAG depletion in both low and high cABC treated FLIm images [Fig. 4(a)]. The untreated control group had an average depleted layer thickness of $\sim 0.1\mathrm{mm}$, whereas low ($1\mathrm{U}/\mathrm{mL}$) and high ($2\mathrm{U}/\mathrm{mL}$) C-ABC concentration treatment resulted in an average depletion layer thickness of 0.3 mm and 0.7 mm, respectively. Average depletion thickness from high cABC treatment group was significantly higher than that of control (untreated) ($p<0.01$) and low cABC ($p<0.01$) treatment group. However, no significant difference was observed in average depletion region thickness between control and low cABC treatment group [Fig. 4(b)].

Fig. 4

(a) Representative cross-sectional FLIm LT and intensity ratio images of cartilage without treatment and cartilage treated with high ($2\mathrm{U}/\mathrm{mL}$) and low ($1\mathrm{U}/\mathrm{mL}$) concentration of cABC. The depleted region is segmented using channel 1 intensity ratio of 0.25. $\text{Scalebar}=1\mathrm{mm}$. (b) Box plot of the thickness of the GAG depleted layer calculated from segmentation result **$p<0.01$ ($n\ge 6$ per treatment group).

4.

Discussion

This study demonstrated that (1) fluorescence LT of articular cartilage varies across its depth; shorter LT values were observed for the superficial zone of cartilage when compared with the middle and deep zones and (2) the depletion of GAG can be detected using FLIm and the level of GAG depletion in articular cartilage can be inferred using fluorescence-based parameters. Both of the above points demonstrate that FLIm can detect biochemical changes in cartilage tissue and has great potential for early detection of cartilage disease.

To our knowledge, this is the first study to map the fluorescence features of native bovine articular cartilage across its full thickness. The cross-sectional imaging protocol allowed us to study changes in FLIm parameters as a function of depth. The comparison of LT distribution obtained from cross-sectional imaging and that from conventional en-face imaging cross-validated the results. Given the complex zonal variation of cell morphology, collagen fiber orientation, and biochemical composition in articular cartilage,⁸ the change of fluorescence LT across the thickness of cartilage is expected. Current results are in agreement with earlier studies conducted on articular cartilage that showed shorter LT for the superficial layer relative to middle and deep zone of the cartilage²⁸ and a previous study that validated en-face FLIm parameters with biochemical assays.²⁹ These results, in conjunction with the fiber-based platform, allow for ease of translation into imaging in humans via augmentation of the current clinical practice of arthroscopy. By incorporating the optical fiber into a conventional viewing scope, this technique would enable straightforward visual evaluation of the integrity of the cartilage surface and inner structures, allow spatial or temporal monitoring during disease development, and allow clinicians to monitor the biochemical changes occurring within the patient.

Apart from the change of fluorescence LT as a function of depth, macrostructures presented in both cross-section (shape of tunnel) and en-face (round shape) exhibit shorter LT when compared with the surrounding cartilage mass. Since cartilage from juvenile bovine condyles was used in this study, these microstructures could be cartilage canals that present only in the early stage of cartilage development.³⁰ Since cartilage canals have different biochemical composition when compared with the collagen-rich cartilage ECM, it is expected that these microstructures exhibit a different LT. This demonstrated that FLIm could be a potential tool for studying basic biochemical properties associated with structural heterogeneities of cartilage as well. To date, much is known about articular cartilage organization and ECM, but less is known about its developmental biology. How articular cartilage grows and matures into a complex, functional, and multifaced structure is still unclear. FLIm could help advance our knowledge in these research areas by monitoring the change of biochemical properties of native articular cartilage during its development and along its depth. The advances will have significant impact for basic science and potential translational value to the design of superior cartilage regeneration and rapier strategies.

The finding that the depletion of GAG significantly altered the fluorescence features of articular cartilage tissue and the change could be detected using our FLIm instrumentation is of critical importance for the research of OA diagnosis, as GAG loss is the first indicator for early OA development. This could enable early diagnosis of OA before clinical symptoms like pain and the appearance of cartilage loss.

We acknowledge that the freeze-thaw cycles of the cartilage tissue before imaging lyse the cells and could alter cartilage fluorescence properties. Thus, the experimental conditions reported in this study might not fully mimic conditions encountered in the clinical application. In a separate study conducted on articular cartilage using the same FLIm instrumentation, we showed that the freezing process results in decreases in channel 1 LT ($\mathrm{\Delta }=0.14$; $p=0.003$) and significant increases in channel 2 LT ($\mathrm{\Delta }=0.11$; $p=0.003$) and channel 3 LT ($\mathrm{\Delta }=0.22$; $p=0.003$) compared to fresh tissue. However, the changes were small compared to those induced by the loss of GAG, which resulted in a significant decrease of fluorescence LT values in channel 2 ($\mathrm{\Delta }=0.44\mathrm{ns}$; $p<0.05$) and channel 3 ($\mathrm{\Delta }=0.75\mathrm{ns}$; $p<0.01$). The small differences in FLIm LT values with cell lysis could be attributed to the very low ($\sim 10\%$ by volume) composition of cells within cartilage tissue.

Interestingly, collagen was more densely packed in the region of GAG depletion post-cABC treatment. This was represented in PSR stained histology as an increase in color saturation in the ROIs. In healthy cartilage, the collagen network is slightly stretched due to the Donnan osmotic pressure caused by GAG content that provides its compressive stiffness.⁸ Thus, the loss of GAG would lead to a slightly denser collagen matrix.

GAG loss resulted in a significant decrease in the intensity ratio of channels 1 and 2, which represents collagen and a significant increase in the intensity ratio of channel 3, which represents GAG. This nonintuitive change could be attributed to the conformational change of collagen structure inside cartilage tissue. It is well known that collagen cross-links strongly fluoresce. The loss of GAG could result in the breakdown of collagen cross-links, which was not quantified in this study. Thus, the loss of GAG very likely resulted in the loss of absolute intensity in all spectral channels, but the loss in channels 1 and 2 was more prominent due to the high quantum yield of collagen and its cross-links. As intensity ratio characterizes the relative intensity contribution of each channel instead of absolute intensity, a higher absolute loss in channels 1 and 2 will result in a decrease in channel 1 and channel 2 intensity ratios and a higher channel 3 intensity ratio.

The decrease of fluorescence LT in channel 2 and channel 3 is a result of the intensity ratio change. Collagen is the dominant fluorophore in cartilage and has the highest fluorescence LT ($\sim 5.5\mathrm{ns}$) of the major constituents.²¹ The decrease of intensity ratio in channel 1 and channel 2 indicates a lower contribution from collagen, which would result in a significant decrease of fluorescence LT in all channels. There is no significant change of fluorescence LT in channel 1 as collagen fluorescence completely dominates this channel. Channel 3 had the largest LT decrease as it sits at the tail of collagen emission spectrum and the collagen contribution is less dominant.

A threshold-based segmentation algorithm could distinguish high levels of GAG depletion in our cABC-treatment group from the control group using one of the many fluorescence parameters. However, intensity measurement is highly sensitive to the excitation and collection geometry. Any change of excitation and collection geometry could lead to significant measurement error. On the other hand, fluorescence LT is robust to the change of measurement conditions. Thus, the effectiveness and robustness of our instrumentation to detect early OA could be further improved by including more FLIm parameters. Nonetheless, we demonstrated that parameters from noninvasive FLIm imaging can successfully detect GAG depletion in articular cartilage.

We employed raster scanning by a translation stage in this study to automate the scanning process. In a clinical setting, the fiber-based interface can be introduced through a standard clinical arthroscope with the FLIm imaging data overlaid on the arthroscope image on the surgical monitor. We have previously demonstrated this concept using a hand scanning probe³¹ for interrogation of breast cancer tumors. Real-time FLIm data were overlaid on conventional white light images of the tissue,³² providing valuable real time feedback to the surgeons. When combined with machine learning algorithms, real-time classification was also possible, providing potential real-time disease discrimination.³¹

We acknowledge that the cross-section imaging approach used in this study is different from an arthroscopy imaging situation. Future en-face imaging will be required to properly assess the performance of FLIm instrumentation for detection of OA and its usefulness in clinical environment. Furthermore, we acknowledge that FLIm is only a surface imaging modality with a penetration depth of $\sim 300\mu \mathrm{m}$ and is not able to provide any structural information regarding the integrity of articular cartilage. However, FLIm can be adapted to tissue interrogation in endoscopic-like configurations,^33,34 thus, it can complement other structural imaging modalities like ultrasound and OCT. This study serves as an important step toward early diagnosis of OA and encourages further investigation into the feasibility of in vivo FLIm arthroscopy.

5.

Conclusion

This study demonstrates that GAG degradation in articular cartilage can be detected by rapid, nondestructive, fiber-based FLIm. This technique could be utilized as a potential tool for basic research of cartilage. In addition, the size of the miniature fiber probe, the clinically compatible high imaging speed, and the potential for multimode imaging, such as FLIm-OCT,³⁵ make it a promising tool for the early diagnosis of OA. This technique has a great potential to facilitate a paradigm shift from palliation of late disease toward OA prevention and early intervention.