Mimicking Loading-Induced Cartilage Self-Heating in Vitro Promotes Matrix Formation in Chondrocyte-Laden Constructs with Different Mechanical Properties

Abstract

Articular cartilage presents a mechanically sensitive tissue. Chondrocytes, the sole cell type residing in the tissue, perceive and react to physical cues as signals that significantly modulate their behavior. Hyaline cartilage is a connective tissue with high dissipative capabilities, able to increase its temperature during daily activities, thus providing a dynamic thermal milieu for the residing chondrocytes. This condition, self-heating, which is still chiefly ignored among the scientific community, adds a new thermal dimension in cartilage mechanobiology. Motivated by the lack of studies exploring this dynamic temperature increase as a potential stimulus in cartilage-engineered constructs, we aimed to elucidate whether loading-induced evolved temperature serves as an independent or complementary regulatory cue for chondrocyte function. In particular, we evaluated the chondrocytes' response to thermal and/or mechanical stimulation in two types of scaffolds exhibiting dissipation levels close to healthy and degenerated articular cartilage. It was found, in both scaffold groups, that the combination of dynamic thermal and mechanical stimuli induced superior effects in the expression of major chondrogenic genes, such as SOX9 and LOXL2, compared to either signal alone. Similar effects were also observed in proteoglycan accumulation over time, along with increased mRNA transcription and synthesis of TRPV4, and for the first time demonstrated in chondrocytes, TREK1 ion channels. Conversely, the chondrogenic response of cells to isolated thermal or mechanical cues was generally scaffoldtype dependent. Nonetheless, the significance of thermal stimulus as a chondro-inductive signal was better supported in both studied groups. Our data indicates that the temperature evolution is necessary for chondrocytes to more effectively perceive and translate applied mechanical loading.

Impact of Molecular Dynamics of Polyrotaxanes on Chondrocytes in Double Network Supramolecular Hydrogels under Physiological Thermomechanical Stimulation

“…While it could be argued that our channel analysis has only been performed in free-swelling constructs and not in stimulated hydrogels, we have previously shown that thermomechanical loading increases the expression of these channels at protein levels as well. 12,26 Hence, we contend the validity of our hypothesis, suggesting that the observed effects likely arise from the interplay between various stimuli and the multifaceted responses of chondrocytes.…”

“…Our previous studies have extensively elucidated the significance and relevance in replicating cartilage self-heating in vitro and demonstrated thoroughly how loading-induced evolved temperature can be harnessed in vitro to accelerate tissue maturation through expression of major structural proteins. 26 Building upon our prior findings where load, heat and hypoxia interactions were studied, herein we aimed to introduce the inherent dynamic nature of the culture environment as an additional parameter in this multifaceted equation. The relevance of including PEG/alpha cyclodextrin-based polyrotaxanes into the robust covalent network stems from their capacity to partially replicate the dynamic interactions found in natural cartilage.…”

Impact of Molecular Dynamics of Polyrotaxanes on Chondrocytes in Double-Network Supramolecular Hydrogels under Physiological Thermomechanical Stimulation

“…In our investigation, the hydrogel formulations were also subjected to bioreactor culture conditions that incorporated thermomechanical signals under a low oxygen tension. Our previous studies have extensively elucidated the significance and relevance in replicating cartilage self-heating in vitro and demonstrated thoroughly how loading-induced evolved temperature can be harnessed in vitro to accelerate tissue maturation through expression of major structural proteins. ,, Building upon our prior findings where load, heat, and hypoxia interactions were studied, herein we aimed to introduce the inherent noncovalent nature of the culture environment as an additional parameter in this multifaceted equation. The relevance of including PEG/alpha cyclodextrin-based polyrotaxanes into the robust covalent network stems from their capacity to partially replicate the dynamic interactions found in natural cartilage. , Through the incorporation of supramolecular polyrataxanes, we aim to mimic the innate noncovalent interactions inherent in cartilage tissue, playing a pivotal role in its mechanical composure and potential signals they transfer to chondrocytes.…”

“…We identified three ion channels, namely, TREK1, TRPV4, and PIEZO1, whose expression was significantly upregulated in the presence of host–guest reversible polyrotaxanes. While it could be argued that our channel analysis has only been performed in free-swelling constructs and not in stimulated hydrogels, we have previously shown that thermomechanical loading increases the expression of these channels at protein levels as well. , Hence, we contend with the validity of our hypothesis, suggesting that the observed effects likely arise from the interplay between various stimuli and the multifaceted responses of chondrocytes.…”

Autologous and Allogeneic Cytotherapies for Large Knee (Osteo)Chondral Defects: Manufacturing Process Benchmarking and Parallel Functional Qualification

“…At the end of the manufacturing phase and following transport/storage, the constructs were evaluated via macroscopic assessment, cell viability assessment, total GAG quantification, and histology for three different transport media (Table 4). [69][70][71]. In order to meet increasing clinical needs, important translational efforts are being allocated toward the optimization of existing ACI protocols or the creation of novel approaches [11,12,[72][73][74][75][76].…”