A Synthetic Thermosensitive Hydrogel for Cartilage Bioprinting and Its Biofunctionalization with Polysaccharides

Abstract

Hydrogels based on triblock copolymers of polyethylene glycol and partially methacrylated poly[N-(2-hydroxypropyl) methacrylamide mono/dilactate] make up an attractive class of biomaterials because of their biodegradability, cytocompatibility, and tunable thermoresponsive and mechanical properties. If these properties are fine-tuned, the hydrogels can be three-dimensionally bioprinted, to generate, for instance, constructs for cartilage repair. This study investigated whether hydrogels based on the polymer mentioned above with a 10% degree of methacrylation (M10P10) support cartilage formation by chondrocytes and whether the incorporation of methacrylated chondroitin sulfate (CSMA) or methacrylated hyaluronic acid (HAMA) can improve the mechanical properties, long-term stability, and printability. Chondrocyte-laden M10P10 hydrogels were cultured for 42 days to evaluate chondrogenesis. M10P10 hydrogels with or without polysaccharides were evaluated for their mechanical properties (before and after UV photo-cross-linking), degradation kinetics, and printability. Extensive cartilage matrix production occurred in M10P10 hydrogels, highlighting their potential for cartilage repair strategies. The incorporation of polysaccharides increased the storage modulus of polymer mixtures and decreased the degradation kinetics in cross-linked hydrogels. Addition of HAMA to M10P10 hydrogels improved printability and resulted in three-dimensional constructs with excellent cell viability. Hence, this novel combination of M10P10 with HAMA forms an interesting class of hydrogels for cartilage bioprinting.

Figure 1

Chemical structure of M₁₀P₁₀ (top) and methacrylated HA (bottom, R = H in equatorial position) or CS (bottom, R = SO₃H in axial position). M₁₀P₁₀ confers thermo-sensitive properties to the gel, whereas the presence of methacrylate groups in both polymers allows UV-mediated chemical cross-linking.

Figure 2

Histology and immunohistochemistry of chondrocytes differentiated in M₁₀P₁₀-based hydrogels (M) with fibrin (fib) as a positive control. From left to right: safranin-O staining, collagen types I, II and VI staining. Scale bars represents 100 µm and is the same for all images of the same staining (column).

Figure 3

Quantitative GAG, DNA, and water measurements for equine chondrocytes encapsulated in M₁₀P₁₀-based hydrogels (M) and fibrin (fib) gels. a) GAG content normalized to DNA for both hydrogels over time. * denotes significant differences compared to day 0; # indicates that the group is significantly higher than the day 0 controls but lower compared to fibrin day 42. $ indicates that the group is significantly higher than the day 0 controls and day 28 fibrin samples but equal to the M hydrogels at days 28 and 42. b, c, d) GAG, DNA and water content normalized to the dry weight (dwt) for M hydrogels over time, respectively. e, f, g) GAG, DNA, and water content normalized to the dry weight (dwt) for fibrin gels over time. ^ significant difference between groups, respectively.

Figure 4

Rheograms of polymer mixtures. G’ (solid line) and G” (dotted line) moduli as a function of temperature, recorded during a temperature sweep experiment from 4 to 50 °C. a) hydrogels based on 18% (w/w) M₁₀P₁₀ (M hydrogels). b) hydrogels based on 14% (w/w) M₁₀P₁₀ and 4% (w/w) CSMA (MCS hydrogels, grey lines) compared with M hydrogels (black lines). c) hydrogels based on 14% (w/w) M₁₀P₁₀ and 0.9% (w/w) HAMA (MHA hydrogels, grey lines) compared with M hydrogels (black lines).

Figure 5

Dynamic mechanical analysis on chemically cross-linked hydrogels. Young’s moduli for hydrogels based on M₁₀P₁₀ (M), hydrogels based on M₁₀P₁₀ and CSMA (MCS) and hydrogels based on M₁₀P₁₀ and HAMA (MHA), measured under unconfined compression (n = 3).

Figure 6

Swelling and degradation profiles for hydrogels based on M₁₀P₁₀ (M), hydrogels based on M₁₀P₁₀ and CSMA (MCS), and hydrogels based on M₁₀P₁₀ and HAMA (MHA) in PBS buffer at 37 °C. Error bars represent the standard deviation of experiments performed in triplicate. SR represents the swelling ratio and was calculated according to equation 1.

Figure 7

3D printed porous constructs based on MHA. a) top view. b) top-side view. c) top-corner view. d) top view showing a homogeneous distribution of encapsulated green fluorescent beads. e) percentage of living chondrocytes in printed and cast (control) constructs for each hydrogel formulation after 1 and 7 days of culture. No statistical differences were observed between hydrogel formulations. Scale bar represents 2 mm.