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

Abstract

Hyaline cartilage, a soft tissue enriched with a dynamic extracellular matrix, manifests as a supramolecular system within load-bearing joints. At the same time, the challenge of cartilage repair through tissue engineering lies in replicating intricate cellular-matrix interactions. This study attempts to investigate chondrocyte responses within double-network supramolecular hybrid hydrogels tailored to mimic the dynamic molecular nature of hyaline cartilage. To this end, we infused noncovalent host-guest polyrotaxanes, by blending α-cyclodextrins as host molecules and polyethylene glycol as guests, into a gelatin-based covalent matrix, thereby enhancing its dynamic characteristics. Subsequently, chondrocytes were seeded into these hydrogels to systematically probe the effects of two concentrations of the introduced polyrotaxanes (instilling different levels of supramolecular dynamism in the hydrogel systems) on the cellular responsiveness. Our findings unveiled an augmented level of cellular mechanosensitivity for supramolecular hydrogels compared to pure covalent-based systems. This is demonstrated by an increased mRNA expression of ion channels (TREK1, TRPV4, and PIEZO1), signaling molecules (SOX9) and matrix-remodeling enzymes (LOXL2). Such outcomes were further elevated upon external application of biomimetic thermomechanical loading, which brought a stark increase in the accumulation of sulfated glycosaminoglycans and collagen. Overall, we found that matrix adaptability plays a pivotal role in modulating chondrocyte responses within double-network supramolecular hydrogels. These findings hold the potential for advancing cartilage engineering within load-bearing joints.

Figure 1

(a) Schematic illustration of elemental motion of polyrotaxanes. (b) ITC experimental curve for the titration of α-CD into PEG_2k solution at 298 K. It shows the heat evolved after each injection at the beginning and saturated after 12–13 injections. (c) Representative ¹H NMR spectra of free α-CD (black line), free PEG_2k (blue line), and α-CD + PEG_2k complexes (red line). (d) Titration curve, which is obtained by the integration of the peaks from Figure 1b, together with a line of best fit, to estimate ΔH, ΔG, TΔS, and the stoichiometry N.

Figure 2

Schematic illustration depicting the various types of hydrogels employed in the study. The polyethylene glycol (PEG) concentration remained consistent across all supramolecular hydrogels, while the alpha-cyclodextrin (α-CD) concentration increased progressively from left to right (a–f). Comparison of the relative expressions of genes of interest, with RPL13a serving as the housekeeping gene.

Figure 3

Histological analysis of neocartilage constructs under free-swelling conditions. (a) Representative images of Alcian Blue staining for sulfated glycosaminoglycan and glycoprotein content (blue). (b) Representative images of Sirius Red staining for general collagen content (red). The intensity of Alcian Blue staining is noticeably higher in supramolecular hydrogels with a higher concentration of alpha-cyclodextrin (α-CD) molecules. Additionally, Sirius Red staining reveals a localized increase as the concentration of α-CD molecules increases. Scale bar: 50 μm, objective 20×.

Figure 4

Interactive effects of mechanobiological cues on chondrocyte ECM deposition. (a) Representative images of Alcian Blue staining for sulfated glycosaminoglycan and glycoprotein content (blue). (b) Representative images of Sirius Red staining for general collagen content (red). (c) Representative images of Masson’s trichrome staining for the total collagen content (blue). (d) Representative images of Safranin-O/Fast green staining for sulfated glycosaminoglycan content are shown for the different types of hydrogels after the last loading cycle was ceased on day 21. Application of biomimetic thermomechanical loading under hypoxia significantly enhanced cartilage-related matrix accumulation, especially in the case of supramolecular hydrogels. Alcian Blue: sulfated GAGs and glycoproteins are stained blue, and the nuclei and cytoplasm pink, Sirius Red: collagen is stained red, and the nuclei dark brown, Masson’s trichrome: collagen is stained blue, and nuclei are stained dark brown, Safranin-O/Fast green: cartilage matrix will be stained orange to red, the nuclei will be stained black, and the background light green. Scale bar: 50 μm, objective 20×. (e) Comparison of the relative expressions of aggrecan (ACAN) and (f) collagen type II (COL2A) after the last loading cycle was ceased, with RPL13a serving as the housekeeping gene.