Mechanical loading inhibits hypertrophy in chondrogenically differentiating hMSCs within a biomimetic hydrogel

Abstract

Three dimensional hydrogels are a promising vehicle for delivery of adult human bone-marrow derived mesenchymal stem cells (hMSCs) for cartilage tissue engineering. One of the challenges with using this cell type is the default pathway is terminal differentiation, a hypertrophic phenotype and precursor to endochondral ossification. We hypothesized that a synthetic hydrogel consisting of extracellular matrix (ECM) analogs derived from cartilage when combined with dynamic loading provides physiochemical cues for achieving a stable chondrogenic phenotype. Hydrogels were formed from crosslinked poly(ethylyene glycol) as the base chemistry and to which (meth)acrylate functionalized ECM analogs of RGD (cell adhesion peptide) and chondroitin sulfate (ChS, a negatively charged glycosaminoglycan) were introduced. Bone-marrow derived hMSCs from three donors were encapsulated in the hydrogels and cultured under free swelling conditions or under dynamic com pressive loading with 2.5 ng/ml TGF-β3. hMSC differentiation was assessed by quantitative PCR and immunohistochemistry. Nine hydrogel formulations were initially screened containing 0, 0.1 or 1mM RGD and 0, 1 or 2wt% ChS. After 21 days, the 1% ChS and 0.1 mM RGD hydrogel had the highest collagen II gene expression, but this was accompanied by high collagen X gene expression. At the protein level, collagen II was detected in all formulations with ECM analogs, but minimally detectable in the hydrogel without ECM analogs. Collagen X protein was present in all formulations. The 0.1 mM RGD and 1% ChS formulation was selected and subjected to five loading regimes: no loading, 5% strain 0.3Hz (1.5%/s), 10% strain 0.3 Hz (3%/s), 5% strain 1 Hz (5%/s), and 10% strain 1Hz (10%/s). After 21 days, ~70-90% of cells stained positive for collagen II protein regardless of the culture condition. On the contrary, only ~20-30% of cells stained positive for collagen X protein under 3 and 5%/s loading conditions, which was accompanied by minimal staining for RunX2. The other culture conditions had more cells staining positive for collagen X (40-60%) and was accompanied by positive staining for RunX2. In summary, a cartilage-like biomimetic hydrogel supports chondrogenesis of hMSCs, but dynamic loading only under select strain rates is able to inhibit hypertrophy.

Characterization of the chondrocyte secretome in photoclickable poly(ethylene glycol) hydrogels

“…Gels were placed in free swelling conditions or in custom bioreactors that were set to apply a compressive strain in a sinusoidal waveform from 0 to 5% at 1 Hz for 1 hour per day. This loading regime was chosen based on previous studies that have shown in PEG hydrogels, the chondrocyte phenotype is maintained and the ECM synthesized is similar or enhanced when compared to free swelling across different donors and cell types (i.e., primary and stem cells) (Aisenbrey and Bryant, 2016; Farnsworth et al, 2013). Medium was replaced every two days for 9 days.…”

“…In addition, the presence of dynamic compressive loading, which is present in the native environment, is an important parameter in cartilage tissue engineering (Butler et al, 2000). Recent studies have identified physiologically relevant loading regimes that support the chondrogenic phenotype and cartilage-specific ECM deposition in PEG hydrogels (Aisenbrey and Bryant, 2016; Farnsworth et al, 2013). Collectively these and other studies support the use of PEG hydrogels for applications in cartilage tissue engineering.…”

“…Second, we aimed to determine whether the secretome varied with different culture conditions, specifically hydrogel crosslink density, which alters the stiffness of the hydrogel, and dynamic compressive loading. In the latter, hydrogel crosslink densities and the regime of dynamic compressive loading were chosen based on prior studies, which have shown promise in promoting cartilage-specific ECM deposition (Aisenbrey and Bryant, 2016; Farnsworth et al, 2013; Mauck et al, 2000; Nicodemus et al, 2011; Park et al, 2011). Photoclickable PEG hydrogels formed from a thiol-norbornene step-growth polymerization were employed due to their rapid polymerizations and ease of use (Fairbanks et al, 2009a; Hoyle and Bowman, 2010; Roberts and Bryant, 2013).…”

Current and novel injectable hydrogels to treat focal chondral lesions: Properties and applicability

“…(16) ChS is the main glycosaminoglycan in cartilage and creates a unique environment that is hyperosmotic and promotes tissue synthesis, especially under dynamic compression. (17) RGD, a cell adhesion peptide, provides a mechanism for cells to sense substrate stiffness and acts as a mechanosensor to cells activating integrin α5β1.…”

Mimetic Hierarchical Approaches for Osteochondral Tissue Engineering

“…It has been detected that moderate amplitude strains (5%) applied at 1 Hz stimulate chondrogenesis of hMSCs and enable stable chondrocyte-like phenotype, while higher strains and lower frequencies have a negative effect on chondrogenesis [95].…”