4.2.5. Polyacrylamide

Polyacrylamide formed from only acrylamide subunits is nonionic. Copolymerizing it with other monomers such as 2-acrylamido-2-methylpropane sulfonate or acrylate forms anionic polyacrylamide, while cationic polyacrylamide could be synthesized upon copolymerization with dimethyl diallyl ammonium. Polyacrylamide substrate is bio-inert; thus, its surface must be conjugating with adhesive ECM proteins to allow for cell attachment [197,198]. Polyacrylamide is widely utilized in literature as a model for investigating the mechanoregulatory role of substrate stiffness combined or uncombined with other parameters in osteogenic differentiation. The stiffness of polyacrylamide hydrogels is commonly modified by altering the concentration of acrylamide monomer or bis-acrylamide crosslinker [62].

Upon seeding human MSCs on 250-Pa polyacrylamide gels coated with a mixture of collagen type 1 and fibronectin, the progression of the cells throughout the cell cycle was prohibited despite the presence of serum. Conversely, the quiescent cells reentered the cell cycle when presented on a stiff polyacrylamide substrate (7.5 kPa). Moreover, the nonproliferative cells revealed an adipogenic differentiation potential upon culturing on 250-Pa gels in adipogenic media or an osteogenic potential into osteoblasts if transferred to a stiff substrate in the presence of osteogenic media [199]. Micropatterned polyacrylamide gels were fabricated with varying stiffness (10 to 40 kPa), using PDMS stamps coated with fibronectin. MSCs cultured on protein-coated gels revealed a stiffness-dependent osteogenic markers' expression (Runx2 and osteopontin) with a maximum expression at 30 kPa [200]. Osteogenic differentiation as revealed by Runx2 expression was upregulated significantly only on collagen I-coated gels with high stiffness (80 kPa), while myogenic differentiation, as ascertained by MyoD1 expression, occurred on all gel–protein coated matrices that had a stiffness of 9 kPa. Peak MyoD1 expression was demonstrated on gels with a modulus of 25 kPa coated with fibronectin. Polyacrylamide hydrogels prepared with variable stiffnesses, ranging from 13 to 68 kPa, through varying the concentrations of bis-acrylamide (0.1%, 0.5%, and 0.7%), showed a difference in the gel morphology. Under scanning electron microscopy, gels with low stiffness (13–16 kPa) appeared flat and non-porous. On the other hand, higher stiffness matrices (48–53 kPa and 62–68 kPa) showed multiple small porosities. Such inherent porosities of polyacrylamide hydrogels could enhance the flow of culture media and better mimic the natural cellular environment, as compared to plastic and glass substrates. Moreover, BMMSCs cultured on 62–68 kPa fibronectin-coated polyacrylamide hydrogels demonstrated a polygonal morphology and revealed an osteogenic phenotype with significantly high levels of ALP, Runx2, and osteopontin [51].

The modulatory effect of extracellular matrix type and density on the mechanotransduction of stem/progenitor cells and the correlated integrin involved in signals translocation were assessed through conjugating each of the four major cell adhesion ECM proteins (fibronectin, collagen I, collagen IV, and laminin) on polyacrylamide hydrogels with tunable stiffness (soft, 3 kPa; and stiff, 38 kPa). The results revealed that increasing ECM ligand density alone can induce YAP nuclear translocation without changing substrate stiffness with a different optimized ligand density. Using antibody-blocking techniques for αvβ3-, α5-, and α2β1-integrins revealed the involvement of αvβ3-, α5-, and α2β1-integrins with fibronectin, while α5-integrin was further associated with collagen type I and IV. On the contrary, laminin was associated with α5- and α2β1-integrins. Moreover, altering ECM type resulted in modulation of human MSC osteogenesis confirmed by quantitative real-time (qRT)-PCR for Runx2 and ALP without changing substrate stiffness [201].

The mechanotransduction role of FAK, α5/β1 integrin and Wnt-signaling pathways mediated by stiff matrices, in regulating osteogenic differentiation of human MSCs cultured on 62–68 kPa fibronectin-coated polyacrylamide hydrogels were further investigated. Throughout osteogenesis, gene and protein expressions of integrin α5/β1 were enhanced, together with the expression of signaling molecules FAK, p-ERK, p-Akt, GSK-3β, p-GSK-3β, and β-catenin. Antibody blocking of integrin α5 significantly downregulated the stiffness-induced expression of osteogenic markers (Runx2, alpha-1 type I collagen, and BGLAP) with associated downregulated expression of ERK, p-ERK, FAK, and β-catenin protein. Reciprocally, GSK-3β, p-GSK-3β, Akt, and p-Akt expressions were upregulated. The presence of the Akt inhibitor Triciribine reduced the expression of p-Akt and p-GSK-3β, whereas Akt, GSK-3β, and β-catenin were unchanged. These results emphasized the role of p-Akt in regulating the expression of p-GSK-3β on 62–68 kPa ECM during osteogenesis [78].

MSCs cultivated on polyacrylamide hydrogels with elasticity (7.0 ± 1.2 and 42.1 ± 3.2 kPa) and coated with type I collagen in osteogenic medium revealed enhanced osteogenic differentiation potential on stiff substrates, with an upregulated expression of Runx2, type I collagen, and osteocalcin genes. On stiff matrices, Western blot analysis revealed an increase in mechanotransducers involved in osteogenic differentiation ROCK, FAK, and ERK1/2, whereas their inhibition resulted in decreased osteogenic markers' expression. Furthermore, α2-integrin was upregulated on stiff matrices during osteogenesis, and its knockdown by siRNA hindered the osteogenic phenotype through FAK, ROCK, and ERK1/2. Therefore, it could be concluded that α2-integrin is involved in osteogenesis mediated by matrix stiffness [62].

Additionally, upon culturing human MSCs on poly acrylamide-co-acrylic acid hydrogels grafted with RGDs, myogenic differentiation occurred at 13–17 kPa, while osteogenic differentiation was revealed at 45–49 kPa stiffness confirmed further with positive protein immunostaining of MyoD, as well as Osx, osteocalcin, and Runx2. Stiffer matrices grafted with BMP-2 mimetic peptide (E = 47.5 kPa) also induced osteoblast lineage commitment, having a similar effect as the ones grafted with RGDs. On the contrary, the osteogenic effect of BMP-2 mimetic peptides on MSCs was inhibited on very soft microenvironments (0.76–3.21 kPa) due to F-actin cytoskeleton reorganization that inhibited BMP-induced smad1/5/8 phosphorylation and subsequent differentiation of the cells into osteoblast-like cells [118].

Umbilical cord (UC) MSCs attained similar behavior upon being cultivated on different stiffness (13–16, 35–38, 48–53, and 62–68 kPa) polyacrylamide gels coated with fibronectin. Quantitative RT-PCR results showed that soft matrices promoted adipogenic differentiation, as evident by upregulated expressions of adipocytic markers (PPARγ and C/EBPα). On the contrary, stiff matrices (48–53 kPa) enhanced the tendency of the cells to differentiate into muscles, as demonstrated by enhanced expression of desmin and MOYG. High stiffness substrates (62–68 kPa) significantly promoted the expression of osteogenic markers, such as Runx2, collagen type I, ALP, and osteocalcin [50].

Moreover, the effect of mechanical loading and biomaterial stiffness on MSCs differentiation was investigated upon cultivating MSCs in osteogenic and adipogenic media on soft (23 ± 0.3 kPa) and stiff (111 ± 2 kPa) polyacrylamide as compared to PDMS (1.5 ± 0.07 MPa) either strained with 8% cyclic strain at 1 Hz or unstrained. Without strain, the expression of ALP was markedly higher on PDMS than on both polyacrylamide types. With 8% cyclic strain, ALP expression was upregulated in all groups, with the highest expression in soft polyacrylamide. Moreover, adipogenesis was the highest on the unstrained soft polyacrylamide, while it was significantly decreased on soft and stiff polyacrylamide when strained [202].
