Gel structure has an impact on pericellular and extracellular matrix deposition, which subsequently alters metabolic activities in chondrocyte-laden PEG hydrogels

Abstract

While designing poly(ethylene glycol) hydrogels with high moduli suitable for in situ placement is attractive for cartilage regeneration, the impact of a tighter crosslinked structure on the organization and deposition of matrix is not fully understood. The objectives for this study were to characterize the composition and spatial organization of neo-matrix as a function of gel crosslinking and study its impact on chondrocytes via anabolic and catabolic gene expressions and catabolic activity. Bovine articular chondrocytes were encapsulated in hydrogels of three crosslinking densities (compressive moduli were 60, 320 and 590 kPa) and cultured for 25 days. Glycosaminoglycan production increased with culture time and was greatest in gels with lowest crosslinking. Collagens II and VI, aggrecan, link protein, and decorin were localized to pericellular regions in all gels, but their presence decreased with increases in gel crosslinking. Collagen II and aggrecan expressions were initially up-regulated in gels with higher crosslinking, but increased similarly up to day 15. Matrix metalloproteinases (MMP)-1 and -13 expressions were elevated (~25-fold) in gels with higher crosslinking throughout the study, while MMP-3 was not affected by gel crosslinking. The presence of aggecan and collagen degradation products confirmed MMP activity. These findings indicate that chondrocytes synthesize the major cartilage components within PEG hydrogels, however, gel structure strongly impacts the composition and spatial organization of the neo-tissue and impacts how chondrocytes respond to their environment, particularly with respect to their catabolic expressions.

Mechanical confinement regulates cartilage matrix formation by chondrocytes

“…2a). This matches the results previously reported in non-degradable elastic hydrogels 20 . Strikingly, as the rate of stress relaxation or creep was enhanced, greater areas of type-II collagen and aggrecan deposition and a more interconnected cartilage matrix were observed in the faster relaxing gels (Fig.…”

“…The elastic moduli of the hydrogels used in these studies ranges from 2 kPa to 100 kPa 12,14,20,21 , which is comparable to the moduli of chondrocyte PCM, ranging from 10 kPa to 75 kPa 29 . Enhanced stiffness in covalently crosslinked PEG based hydrogels, found to exhibit minimal stress relaxation here, restricted production of sGAG and collagens, distribution of cartilage matrix, and proliferation of chondrocytes 20,21 . However, our work revealed that the impact of stiffness was dependent on hydrogel relaxation.…”

“…As previous work has implicated hydrogel stiffness in mediating cartilage matrix formation 20,21,30 , we next examined how altered hydrogel stiffness regulated the effect of relaxation. In hydrogels with an initial elastic modulus of 20 kPa, trends similar to those in 3 kPa gels were observed, with faster relaxation leading to greater areas of type-II collagen and aggrecan deposition as well as higher quantities of secreted collagen and sGAG (Supplementary Fig.…”

“…Recent studies have found that viscoelastic hydrogels, in which stresses are relaxed over time, promote spreading and proliferation of adherent cells, and formation of an interconnected bone matrix by osteogenically differentiated MSCs in 3D culture 18,19 . While there has been extensive study of the impact of hydrogel stiffness or elasticity on cartilage matrix formation by chondrocytes 20–22 , the impact of hydrogel stress relaxation, related to viscosity and creep, on chondrocytes remains unknown. Here we test whether viscoelastic hydrogels that exhibit fast stress relaxation could provide a microenvironment that is more conducive to cartilage matrix formation by chondrocytes.…”

The pericellular environment regulates cytoskeletal development and the differentiation of mesenchymal stem cells and determines their response to hydrostatic pressure

“…One potential explanation for this is that diffusivity of biomolecules (such as TGF-β3) would be lower in the stiffer, denser 4 % hydrogels; however, these hydrogels are still 96 % fluid and are therefore not expected to significantly inhibit biomolecule diffusivity. While MSCs cannot directly adhere to agarose, and hence initially are unlikely to be able to sense their local stiffness, they rapidly synthesise fibronectin and other extracellular matrix components in hydrogel culture (Nicodemus et al, 2011;Parekh et al, 2011) to which they can adhere, which may provide them with a mechanism through which they can sense the stiffness of the surrounding hydrogel.…”