Abstract
Summary Statement
We have developed 3D combinatorial hydrogels containing cartilage extracellular matrix (ECM) proteins for modulating chondrogenesis of adipose-derived stromal cells. Our platform allows independently tunable biochemical and mechanical properties, which may provide a valuable tool for elucidating how ECM biochemical cues interact with matrix stiffness to regulate stem cell chondrogenesis.
Introduction
Adipose-derived stromal cells (ADSC) hold great promise for cartilage repair given their relative abundance and ease of isolation. Biomaterials can serve as artificial niche to direct chondrogenesis of ADSCs, and extracellular matrix (ECM) protein-based scaffolds are highly biomimetic. However, incorporating ECM molecules into hydrogel network often lead to simultaneous changes in both biochemical ligand density and matrix stiffness. This makes it difficult to understand how various niche signals interact together to regulate ADSC fate. To overcome these limitations, the goal of this study is to develop an ECM-containing hydrogel platform with independently tunable biochemical and mechanical cues for modulating ADSC chondrogenesis in 3D. We hypothesise that decreasing the degree of crosslinking of ECM molecules may allow their incorporation without affecting the matrix stiffness. The effects of interactive signaling between ECM molecules and matrix stiffness on ADSC chondrogenesis in 3D was then examined using this platform
Methods
Three types of cartilage-specific extracellular matrix proteins (ECM) - chondroitin sulfate (CS), hyaluronic acid (HA) and heparan sulfate (HS) were chemically incorporated into hydrogel network via methacrylation. ECM proteins were modified with methacrylate end groups at varying degrees of methacrylation to allow minimal influence on matrix mechanical properties. To vary biochemical cues within the hydrogels, CS, HA and HS were incorporated at varying concentrations (0.5, 1.25, 2.5 and 5% w/v). To examine the stability of ECM molecules with varying degrees of methacrylation within the hydrogel network, ECM molecules are fluorescently labeled and incorporated within hydrogels. Leaching was monitored by measuring fluorescence over time. To vary mechanical property of the hydrogels, poly-(ethylene glycol) dimethylacrylate (PEGDMA) (4.6kDa) was incorporated at varying concentrations (5, 10, and 15% w/v). Based on the leaching test and mechanical test, ECM molecules with the optimal degree of methacrylation that allows stable interlocking without changing matrix stiffness were chosen for further studies. A total of 39 combinatorial hydrogel compositions were examined. Human ADSCs were encapsulated in combinatorial hydrogels and supplied chondrogenic medium for 21 days. Outcome was analyzed by quantifying chondrogenic gene expression (Aggrecan and Collagen II) and immunofluorescent staining.
Results
Mechanical testing showed that hydrogel stiffness was controlled solely by varying the concentration of mechanical blocks (PEGDMA), and was not influenced by varying the ECM concentration within the range tested (up to 5% w/v). Increasing PEGDMA concentration from 5% to 15%(w/v) produced hydrogels with stiffness from 3–100kPa. Results from the leaching test confirmed that majority of the ECM molecules were stably crosslinked within the hydrogel network. Gene expression results showed that biochemical and mechanical signals interacted in a non-linear manner. At lower concentrations of biochemical cues, increasing mechanical stiffness promoted chondrogenesis. However, at higher concentrations of biochemical cues, biochemical cues played a more important role on regulating chondrogenesis. In soft matrix (5% PEGDMA), both HA and HS increased chondrogenesis in a dose-dependent manner
Discussion/ Conclusion
Here we report an ECM-containing hydrogel platform with independently tunable mechanical and biochemical cues. We identified optimised degree of methacrylation of ECM molecules that allowed their stable incorporation via covalent bonding, without affecting the matrix stiffness. This platform may provide a useful tool to facilitate elucidating how interactive niche signals regulate stem cells fate in 3D, and identify optimal scaffold compositions to promote musculoskeletal tissue differentiation.
