Contrast enhanced computed tomography for real-time quantification of glycosaminoglycans in cartilage tissue engineered constructs

Abstract

Tissue engineering and regenerative medicine are two therapeutic strategies to treat, and to potentially cure, diseases affecting cartilaginous tissues, such as osteoarthritis and cartilage defects. Insights into the processes occurring during regeneration are essential to steer and inform development of the envisaged regenerative strategy, however tools are needed for longitudinal and quantitative monitoring of cartilage matrix components. In this study, we introduce a contrast-enhanced computed tomography (CECT)-based method using a cationic iodinated contrast agent (CA4+) for longitudinal quantification of glycosaminoglycans (GAG) in cartilage-engineered constructs. CA4+ concentration and scanning protocols were first optimized to ensure no cytotoxicity and a facile procedure with minimal radiation dose. Chondrocyte and mesenchymal stem cell pellets, containing different GAG content were generated and exposed to CA4+. The CA4+ content in the pellets, as determined by micro computed tomography, was plotted against GAG content, as measured by 1,9-dimethylmethylene blue analysis, and showed a high linear correlation. The established equation was used for longitudinal measurements of GAG content over 28 days of pellet culture. Importantly, this method did not adversely affect cell viability or chondrogenesis. Additionally, the CA4+ distribution accurately matched safranin-O staining on histological sections. Hence, we show proof-of-concept for the application of CECT, utilizing a positively charged contrast agent, for longitudinal and quantitative imaging of GAG distribution in cartilage tissue-engineered constructs.

Statement of Significance

Tissue engineering and regenerative medicine are promising therapeutic strategies for different joint pathologies such as cartilage defects or osteoarthritis. Currently, in vitro assessment on the quality and composition of the engineered cartilage mainly relies on destructive methods. Therefore, there is a need for the development of techniques that allow for longitudinal and quantitative imaging and monitoring of cartilage-engineered constructs. This work harnesses the electrostatic interactions between the negatively-charged glycosaminoglycans (GAGs) and a positively-charged contrast agent for longitudinal and non-destructive quantification of GAGs, providing valuable insight on GAG development and distribution in cartilage engineered constructs. Such technique can advance the development of regenerative strategies, not only by allowing continuous monitoring but also by serving as a pre-implantation screening tool.

Introduction

Joint disorders are a major cause of morbidity and disability worldwide, and represent both significant healthcare and socio-economical burdens [1]. Osteoarthritis (OA) and joint trauma, which adversely affect articular cartilage, ultimately lead to its degradation over time [2], [3]. The extracellular matrix (ECM) of cartilage is mainly composed of proteoglycans, collagen II, and water [2]. The pathogenesis of degeneration is still poorly understood, however degradation is accompanied by breakdown of proteoglycans, which inherently induces a reduction in the mechanical properties and poorer functional performance of the tissue [2]. Currently, disease modifying therapies remain unavailable for OA and treatments are limited to pain relief and palliative care at early stages, and joint prosthesis at the end-stage [2], [4]. Hence, there is a need for new treatment modalities that promote tissue repair and regeneration. In this context, tissue engineering and regenerative medicine are of interest as therapeutic approaches [4], [5], which employ the use of cells, biomaterial scaffolds, and/or stimulatory factors to ultimately produce cartilaginous-like tissue [5]. Currently, most of the available techniques to assess the effects of these factors on matrix production and tissue quality are destructive. Usually, engineered-tissues are harvested at mid or endpoints and subjected to biochemical assays such as the 1,9-dimethylmethylene blue (DMMB) and hydroxyproline assays for quantification of total glycosaminoglycans (GAGs) and collagen content, respectively [6], [7]. In addition, constructs can be processed for histology and immunohistochemistry (IHC), which only provide a two-dimensional and qualitative assessment of matrix components and tissue quality [8], [9]. However, the detailed understanding of the regeneration process over time is crucial for achieving full regenerative potency in vitro and in vivo. Therefore, it is of significant importance to develop non-invasive and non-destructive imaging techniques for real-time, three-dimensional (3D), and quantitative monitoring of cartilage tissue-engineered constructs. Such techniques will enable monitoring of in vitro regeneration over time, evaluation of ECM components, optimization of chondrogenic activity, and, ultimately, screening to identify the best performing tissue constructs before implantation.

Efforts are ongoing to develop such tools and techniques. For example, ultrasound was used as a standalone procedure or in combination with fluorescence techniques to assess both matrix composition and mechanical properties in cartilage tissue-engineered constructs [10], [11], [12], [13], [14]. More recently, a set of reporter genes was described for transfection of MSCs as a monitoring tool for real-time characterization of the chondrogenic differentiation process [15]. Also dielectric impedance spectroscopy was proposed as a label-free and non-destructive method to evaluate cellular viability and survival during and after biofabrication processes [16]. Despite these recent advances, most of the described techniques are qualitative, do not quantify ECM components, or lack the resolution to assess the 3D distribution of the matrix components.

Contrast-enhanced computed tomographic (CECT) imaging is a rapid and readily available imaging modality used to study many different tissues [17], namely tissues with low X-ray attenuation to include articular cartilage [18], [19], [20], [21], [22], meniscus [23], [24], [25], intervertebral disc [26], [27], [28], and xiphoid cartilage [29]. Due to the compositional differences among these tissues, contrast agent diffusion and flux will vary [20], [24]. While GAGs are mainly responsible for electrostatic interactions, collagen fibers will drive steric hindrance [24]. CECT provides unique high-resolution 3D information and quantification on composition and distribution of crucial constituents within articular cartilage. Charge-driven transport of negatively or positively charged iodinated contrast agents (i.e., ioxaglate and CA4+, respectively) provides more efficient imaging of GAGs with greater sensitivity [30], [31], [32], [33]. Due to the anionic fixed charge of cartilage ECM, anionic contrast agents inversely correlate with GAG content, while positively-charged contrast agents display a positive correlation with GAG content with considerably higher sensitivity [18], [20], [28], [30], [34], [35], [36], [37], [38]. Hence, we hypothesize that CA4+-based CECT will allow for longitudinal imaging and GAG quantification in cartilage tissue-engineered constructs. In this work, we propose a CECT-based approach as a high-resolution 3D “histology” technique for real-time spatiotemporal quantification of total GAG content in tissue-engineered constructs, which potentially replaces the currently available destructive assays.