The influence of hyaluronic acid hydrogel crosslinking density and macromolecular diffusivity on human MSC chondrogenesis and hypertrophy

Abstract

Hyaluronic acid (HA) hydrogels formed via photocrosslinking provide stable 3D hydrogel environments that support the chondrogenesis of mesenchymal stem cells (MSCs). Crosslinking density has a significant impact on the physical properties of hydrogels, including their mechanical stiffness and macromolecular diffusivity. Variations in the HA hydrogel crosslinking density can be obtained by either changes in the HA macromer concentration (1, 3, or 5% w/v at 15 min exposure) or the extent of reaction through light exposure time (5% w/v at 5, 10, or 15 min). In this work, increased crosslinking by either method resulted in an overall decrease in cartilage matrix content and more restricted matrix distribution. Increased crosslinking also promoted hypertrophic differentiation of the chondrogenically induced MSCs, resulting in more matrix calcification in vitro. For example, type X collagen expression in the high crosslinking density 5% 15 min group was ~156 and 285% higher when compared to the low crosslinking density 1% 15 min and 5% 5 min groups on day 42, respectively. Supplementation with inhibitors of the small GTPase pathway involved in cytoskeletal tension or myosin II had no effect on hypertrophic differentiation and matrix calcification, indicating that the differential response is unlikely to be related to force-sensing mechanotransduction mechanisms. When implanted subcutaneously in nude mice, higher crosslinking density again resulted in reduced cartilage matrix content, restricted matrix distribution, and increased matrix calcification. This study demonstrates that hydrogel properties mediated through alterations in crosslinking density must be considered in the context of the hypertrophic differentiation of chondrogenically induced MSCs.

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

“…In agreement with previous studies (Bian et al, 2013;Erickson et al, 2009), a denser, presumably stiffer, PCM also develops in the higher concentration hydrogels to which the MSCs can adhere and sense. In addition to a stiffer micro-environment for MSCs in the 4 % hydrogels, it is also reasonable to assume that the more developed PCM in these constructs leads to an increase in the number of integrin binding sites per cell.…”

“…Irrespective of hydrogel stiffness, this permissive medium did not support chondrogenesis of MSCs. As seen previously, when maintained in a medium supplemented with TGF-β3, cartilage matrix production was inhibited in stiffer hydrogels (Bian et al, 2013;Erickson et al, 2009). 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.…”

Cyclic hydrostatic pressure promotes a stable cartilage phenotype and enhances the functional development of cartilaginous grafts engineered using multipotent stromal cells isolated from bone marrow and infrapatellar fat pad

“…Several studies have also indicated that HP can trigger the release of intracellular calcium stores (Browning et al, 2004;Mizuno, 2005), but further investigation will be necessary to determine whether such factors regulate the onset of hypertrophy in BMSCs. What has been demonstrated is that a stiffer substrate promotes a more endochondral phenotype in BMSCs (Bian et al, 2013). Since the pericellular environment will become stiffer as ECM content increases with time in culture, this may contribute to the development of an endochondral phenotype in BMSC constructs.…”

Cell morphology and focal adhesion location alters internal cell stress

“…Rigid no-slip boundary conditions were assigned along FA regions (u ¼ 0, with u denoting nodal displacement), while nodes along the untied cell-substrate interface were assigned a rigid free-slip boundary condition (u Án ¼ 0, n  @u=@n ¼ 0). Approximately 150 000 (dendritic model) or 300 000 (spread model) quadratic tetrahedral C3D10R elements were used, with elements ranging in size from 16 000 nm 3 at the FA sites to 10 mm 3 at the outermost edge of the substrate were used for all models. Mesh convergence data were generated to verify the number of elements used.…”

“…Extracellular mechanical cues, such as differences in passive substrate stiffness, externally applied mechanical strain and fluid-flow induced shear stress, have been shown to affect many aspects of cell behaviour, including migration, proliferation and differentiation [1][2][3][4][5]. The internal machinery of the cell, consisting of tensile (actin) and compressive (microtubule) elements, and FA attachment complexes play an important role in the translation of extracellular forces and alter fundamental cell behaviours, such as viability and migration, through the generation of intracellular stress [6].…”