Decorin regulates cartilage pericellular matrix micromechanobiology

Highlights

• Decorin regulates the aggrecan network integrity and micromechanics of cartilage pericellular matrix.

• The highly negative charged osmotic microenvironment of pericellular matrix is required for normal chondrocyte mechanotransduction in situ.

• Decorin affects the intracellular calcium signaling of chondrocytes by mediating the aggrecan-endowed osmotic microenvironment of pericellular matrix.

• The impact of decorin loss on the disruption of chondrocyte mechanobiology is increasingly aggravated during maturation.

Abstract

In cartilage tissue engineering, one key challenge is for regenerative tissue to recapitulate the biomechanical functions of native cartilage while maintaining normal mechanosensitive activities of chondrocytes. Thus, it is imperative to discern the micromechanobiological functions of the pericellular matrix, the ~ 2–4 µm-thick domain that is in immediate contact with chondrocytes. In this study, we discovered that decorin, a small leucine-rich proteoglycan, is a key determinant of cartilage pericellular matrix micromechanics and chondrocyte mechanotransduction in vivo. The pericellular matrix of decorin-null murine cartilage developed reduced content of aggrecan, the major chondroitin sulfate proteoglycan of cartilage and a mild increase in collagen II fibril diameter vis-à-vis wild-type controls. As a result, decorin-null pericellular matrix showed a significant reduction in micromodulus, which became progressively more pronounced with maturation. In alignment with the defects of pericellular matrix, decorin-null chondrocytes exhibited decreased intracellular calcium activities, [Ca²⁺]_i, in both physiologic and osmotically evoked fluidic environments in situ, illustrating impaired chondrocyte mechanotransduction. Next, we compared [Ca²⁺]_i activities of wild-type and decorin-null chondrocytes following enzymatic removal of chondroitin sulfate glycosaminoglycans. The results showed that decorin mediates chondrocyte mechanotransduction primarily through regulating the integrity of aggrecan network, and thus, aggrecan-endowed negative charge microenvironment in the pericellular matrix. Collectively, our results provide robust genetic and biomechanical evidence that decorin is an essential constituent of the native cartilage matrix, and suggest that modulating decorin activities could improve cartilage regeneration.

Introduction

A key paradox in cartilage regeneration is that while a soft mechanical environment is required for maintaining chondrocyte phenotype [1], a much higher modulus is needed for engineered products to recapitulate the function of native tissue [2]. When cultured in vitro, chondrocytes are encapsulated in soft hydrogels with modulus ~ 10 kPa or less to maintain cell viability and prevent de-differentiation [1]. In this environment, while chondrocytes can synthesize major cartilage extracellular matrix (ECM) constituents, namely type II collagen and aggrecan, these molecules do not assemble into the hierarchical structure of the native ECM, thereby failing to fully restore the biomechanical properties of native tissue [3]. In vivo, chondrocytes reside within the pericellular matrix (PCM), a structurally distinctive, ~ 2–4 μm-thick cell-ECM intermediary [4]. The PCM is pivotal in transmitting biomechanical, biophysical and biological signals between the ECM and cells [5, 6], and is where the initial assembly of newly synthesized matrix molecules takes place [7,8]. In healthy human cartilage, the modulus of PCM is ~ 50 kPa [9], much higher than that of the canonical hydrogel environment [1]. Despite being surrounded by a much stiffer matrix, residing chondrocytes can sustain their normal metabolic activities during development and maintenance in vivo. In osteoarthritis (OA), degeneration of the PCM is one of the earliest events upon disease initiation, leading to aberrant chondrocyte mechanotransduction, which contributes to the vicious loop of irreversible cartilage degeneration [9], [10], [11]. Understanding the biomechanical and biophysical characteristics of the native PCM will provide a much needed benchmark for engineered tissues to better recapitulate the native microniche of chondrocytes [12]. In addition, it is increasingly evident that changes in the ECM are the driving force of most human diseases, both congenital and acquired [13], [14], [15]. Knowledge about cartilage PCM could thus provide new insights into the roles of immediate cell microniche in other diseases as well [16], [17], [18].

Over the past decades, there have been significant advances in understanding the roles of individual PCM biomolecules in cartilage health and disease [19], including type VI collagen [20], [21], [22], perlecan [23], [24], [25], biglycan [26,27] and matrilins [28], [29], [30], [31]. Despite these efforts, the assembly, structure and mechanobiological functions of PCM remain poorly understood [5]. It is unclear how the native PCM maintains normal chondrocyte activities despite being much stiffer than the in vitro chondrogenic microenvironment. One distinctive feature of the native PCM is the preferential localization of specific proteoglycans [32]. In particular, aggrecan, the major proteoglycan of cartilage, is more concentrated in the PCM [33], where it undergoes faster turnover [34], and with newly synthesized aggrecan mainly localized there [35]. Aggrecan has a bottle-brush architecture, consisting of ~ 400 nm-long core protein decorated with > 100 densely packed, ~ 40 nm-long chondroitin sulfate glycosaminoglycan (CS-GAG) side chains. In cartilage, aggrecan contributes to > 90% of CS-GAGs and total fixed charges [19], and endows the matrix with its highly negatively charged environment [36]. Although the contribution of aggrecan to cartilage tissue-level biomechanics has been well documented [37], it is unclear whether or how aggrecan and its fixed negative charges impact the pericellular microenvironment and chondrocyte mechanosensing in vivo. It is also unclear how the structural integrity of aggrecan is maintained in the PCM, for that the primary assembly mechanism of aggrecan network, the link protein-assisted aggrecan-hyaluronan (HA) aggregation [38], does not fully address the diversity or changes in the retention of aggrecan at different development stages and disease states, or the preferential distribution of aggrecan in the PCM [35].

Decorin, a small leucine-rich proteoglycan, could play an important role in regulating the integrity of aggrecan in the PCM. Our recent studies showed that in decorin-null (Dcn^−/−) mice, loss of decorin leads to markedly reduced retention of aggrecan in the ECM, resulting in impaired cartilage biomechanical properties [39] and increased susceptibility to surgery-induced OA [40]. We further showed that decorin primarily functions as a “physical linker” to increase the molecular adhesion between aggrecan-aggrecan and aggrecan-collagen II fibrils, thereby strengthening the integration of aggrecan networks without directly affecting chondrocyte biology or aggrecan biosynthesis [39]. In adult cartilage ECM, decorin and aggrecan are present in both the PCM and the territorial/interterritorial ECM (T/IT-ECM) that is further removed from cells [39]. We thus hypothesize that decorin is required for maintaining the integrity of aggrecan networks in the PCM. We tested this hypothesis by studying the nanostructural and micromechanical phenotype of Dcn^−/− cartilage PCM. We further queried if loss of decorin disrupts chondrocyte mechanotransduction by assessing the intracellular calcium signaling activities, [Ca²⁺]_i, in situ, which are one of the earliest, fundamental cell responses to mechanical stimuli [41]. Then, by studying [Ca²⁺]_i signaling under enzymatic removal of CS-GAGs, we tested if the impact of decorin on chondrocytes is manifested through its regulation of aggrecan assembly in the PCM. Our findings suggest that the aggrecan-rich, highly negatively charged PCM microenvironment is essential for maintaining normal chondrocyte mechanosensitive activities, and decorin plays an important role in regulating the integrity of aggrecan in this critical microdomain.