Introduction: Matrix stiffness has been shown to play an important role in regulating stem cell differentiation in 2D, yet how matrix stiffness influence stem cell chondrogenesis in 3D remain largely unknown. Hydrogel with varying stiffness could be achieved using materials with different crosslinking mechanisms and degradation profiles. PEGDMA and methacrylated chondroitin sulfate (CS) are two most commonly used photopolymerizable hydrogels for guiding chondrogenesis of MSCs for cartilage repair. While varying concentration of both materials can lead to simultaneous changes in hydrogel stiffness, PEG is bioinert and non-degradable, and CS is a natural component of cartilage and degradable by cell-secreted enzymes. The goal of this study is to examine the effects of matrix stiffness on modulating chondrogenesis of mesenchymal stem cells in 3D, and investigate the effects of increasing hydrogel stiffness by adding additional non-degradable PEGDMA or increasing methacrylation of CS, on MSC fate.
Materials and Methods: Hydrogels with tunable stiffness were fabricated by either 1) increasing degree of methacrylation of CS, thereby increasing crosslinking density (using regular CS-MA or highly methacrylated CS (hCS-MA) or 2) adding non-degradable PEGDMA. A total of six hydrogel formulations were examined (Table 1). Parameters to vary include two CS concentrations (3% and 5% w/v), two degree of methacylation of CS (normal or high). To match the stiffness of 3% or 5% hCS-MA, PEGDMA were added into 3% or 5% normal CS-MA. Passage 6 human MSCs were encapsulated in all hydrogel groups and cultured for 21 days in chondrogenic medium with 10ng/ml TGF-β3. Outcomes were analyzed via gene expression, biochemical assays (DNA, sGAG, hydroxyproline) and histology.
Results: Mechanical testing confirmed that hCS-MA increased hydrogel stiffness compared to regular CS-MA hydrogels at comparable CS concentrations (3% or 5%). Adding additional PEG allowed achieving comparable hydrogel stiffness with hCS-MA while keeping CS concentration constant (Fig 1). In 5% CS groups, in which adding PEG (group 6) led to substantial down-regulation of cartilage markers (Agg and Col II) compared to hCS-MA (Fig 2A). The opposite trend was observed in 3% CS groups. Increasing hydrogel stiffness using different mechanisms invariably led to higher MMP 13 expression (groups 2, 3, 5, 6) (Fig 2A). Biochemical analyses (DNA, sGAG, collagen) showed that MSCs encapsulated in hCS-MA hydrogels produced more sGAG and collagen as compared to their corresponding PEG containing hydrogels with comparable stiffness (Fig 2B). Safranin-O and collagen II staining showed that MSCs in CS-MA and hCS-MA hydrogels were able to completely remodel their hydrogels and form interconnected neocartilage matrix (groups 1,2 ,4, 5) (Fig 2D, E), while PEG-containing hydrogels restricted cell proliferation (Fig 2B) and new matrix production was limited to pericellular regions only. hCS-MA containing hydrogels also led to higher mechanical modulus as compared to the acellular control after 21 days (Fig 2C).
Discussion: Our results showed that the effects of hydrogel stiffness on MSC chondrogenesis is highly dependent upon the mechanisms of crosslinking and biochemical cues. Increasing hydrogel stiffness by increasing methacrylation of fully degradable CS hydrogels were more superior to PEGDMA containing hydrogels in guiding MSC proliferation, chondrogenesis and neocartilage deposition in an interconnected manner leading to superior mechanical property restoration.
NIH R01DE024772 (F.Y); California Institute for Regenerative Medicine (Grant #TR3-05569) (F. Y.),; National Science Foundation CAREER award program (CBET-1351289, F.Y.); Stanford Chem-H Institute seed grant (F. Y.); Stanford Bio-X Interdisplinary program award (F. Y.); Stanford Child Health Research Faculty Scholar award (F.Y.); A*STAR PhD fellowship (T.W.)