Strategy Based on Michael Addition Reaction for the Development of Bioinspired Multilayered and Multiphasic 3D Constructs

Abstract

The high incidence of osteochondral defects has increased the interest in the development of improved repairing alternatives, with tissue engineering being considered a promising approach. The hierarchical, complex structure of osteochondral tissue requires the design of a biomimetic multilayered scaffold. Here, a multilayered and multiphasic 3D macroporous structure was achieved at subzero temperature by the Michael addition reaction of amino functionalities of collagen with acryloyl groups of a bifunctionalized poly(ε-caprolactone). This green approach has been successfully applied to crosslink layers of different composition, both for their efficient sequential formation and connection. Polyethylenimine functionalized nano-hydroxyapatite (nHAp_LPEI) was added to the bottom layer. The resulting hybrid cryogels were characterized by morphology, equilibrium swelling ratios, compressive strength analysis, and MTS assay. They presented good stability, integrity, and biocompatibility. The results revealed that the properties of the prepared constructs may be tuned by varying the composition, number, and thickness of the layers. The Young modulus values were between 3.5 ± 0.02 and 10.5 ± 0.6 kPa for the component layers, while for the multilayered structures they were more than 7.3 ± 0.2 kPa. The equilibrium swelling ratio varied between 4.6 and 14.2, with a value of ~10.5 for the trilayered structure, correlated with the mean pore sizes (74-230 µm).

Keywords: aza-Michael addition; biocomposite; cryogelation; multilayered construct; tissue engineering.

Scheme 1

Schematic representation of the cryogels’ synthesis strategy.

Figure 1

Characteristic micrographs (typical microstructures) for the CP7 sample prepared under different conditions: (A) 37 °C, final dispersion concentration 1.25%, (B) −12 °C, final dispersion concentration 1.25%, and (C) −12 °C, concentration of reaction medium 2.4%.

Figure 2

FTIR spectra of commercial collagen and of the CP7 sample (Entry 2, Table 1).

Figure 3

Swelling behavior: effect of the synthesis conditions for the CP7 sample (Entry 2, Table 1) obtained at different temperatures and feed concentrations: (A) −12 °C, c—2.4%; (B) −12 °C, c—1.25%; (C) 37 °C, c—1.25%.

Figure 4

Real-time imaging of sample cryogel CP7 (Entry 2, Table 1) during the compression test.

Figure 5

Cyclic compressive properties of sample CP7 (Entry 2, Table 1).

Figure 6

Schematic representation of the typical compositional (wt.%) variation in a trilayered construct (sample TL1).

Figure 7

Typical FTIR spectra of the included construct layers of different compositions.

Figure 8

Representative SEM images for the prepared cryogels (mono-, bi-, and trilayered): (A) sample 2/CP7, (B) and (D) bilayered constructs at the interface CH5P15HAp20/CH5P10HAp5, (C) sample 3/CH5P15HAp20, (E) TL1 trilayered sample (CH10P20HAp20/CH7P10HAp5/CH5P10), and (F) TL2 trilayered sample (CH7P10/CH7P10HAp5).

Figure 9

Swelling kinetics of the prepared cryogel constructs for different composition, morphology, and architecture: (A) sample 7 (CH5P10), (B) TL1 trilayered sample (CH10P20HAp20/CH7P10HAp5/CH5P10), (C) bilayered structure comprising formulations 5 and 6 (CH10P20HAp20/CH7P10HAp5), and (D) sample 5 (CH10P20HAp20).

Figure 10

Cell viability of normal fibroblasts exposed to different samples’ extracts at various concentrations in complete cell culture medium (2.5 mg/mL, 5 mg/mL, 7.5 mg/mL, and 10 mg/mL) for 24 h.