Osteochondral Regeneration with 3D-Printed Biodegradable High-Strength Supramolecular Polymer Reinforced-Gelatin Hydrogel Scaffolds

Abstract

Biomacromolecules with poor mechanical properties cannot satisfy the stringent requirement for load-bearing as bioscaffolds. Herein, a biodegradable high-strength supramolecular polymer strengthened hydrogel composed of cleavable poly(N-acryloyl 2-glycine) (PACG) and methacrylated gelatin (GelMA) (PACG-GelMA) is successfully constructed by photo-initiated polymerization. Introducing hydrogen bond-strengthened PACG contributes to a significant increase in the mechanical strengths of gelatin hydrogel with a high tensile strength (up to 1.1 MPa), outstanding compressive strength (up to 12.4 MPa), large Young's modulus (up to 320 kPa), and high compression modulus (up to 837 kPa). In turn, the GelMA chemical crosslinking could stabilize the temporary PACG network, showing tunable biodegradability by adjusting ACG/GelMA ratios. Further, a biohybrid gradient scaffold consisting of top layer of PACG-GelMA hydrogel-Mn²⁺ and bottom layer of PACG-GelMA hydrogel-bioactive glass is fabricated for repair of osteochondral defects by a 3D printing technique. In vitro biological experiments demonstrate that the biohybrid gradient hydrogel scaffold not only supports cell attachment and spreading but also enhances gene expression of chondrogenic-related and osteogenic-related differentiation of human bone marrow stem cells. Around 12 weeks after in vivo implantation, the biohybrid gradient hydrogel scaffold significantly facilitates concurrent regeneration of cartilage and subchondral bone in a rat model.

Keywords: 3D printing; biohybrid gradient scaffolds; high strength; osteochondral regeneration; supramolecular polymers.

Scheme 1

Schematic illustration of 3D printing of the biohybrid gradient scaffolds for repair of osteochondral defect. A) The compositions of bioink A and bioink B, and 3D‐bioprinting method of the biohybrid gradient scaffolds assisted with a low‐temperature receiver; B) formation of stable hydrogel scaffold after UV light‐initiated polymerization and main hydrogen bonding interactions in the PACG‐GelMA network; C) the repair of osteochondral defects treated with the biohybrid gradient PACG‐GelMA hydrogel scaffold with Mn²⁺ and BG being respectively loaded on the top layers and bottom layers in animal experiment.

Figure 1

A–C) Tensile stress–strain curves and D–F) compressive stress–strain curves of the PACG‐GelMA hydrogels with varied initial concentrations of ACG and GelMA in deionized water.

Figure 2

In vitro degradation behaviors of PACGX‐GelMAY hydrogels with varied initial concentrations of ACG and GelMA in collagenase solution.

Figure 3

A,B) Influences of the concentration of GelMA and ACG, and addition of BG on the rheological properties of the hydrogels. Variation in dynamic storage moduli G′ and loss moduli G″ as a function of temperature in a temperature amplitude sweep test, where the cross‐over points between the gel and sol state (G′ = G″) represent gel‐sol transition temperature; C,D) variation of viscosity as a function of temperature; E,F) viscosity measurement of different bioinks in a shear rate sweep from 0.1 to 500 s⁻¹, indicative of shear‐thinning behavior.

Figure 4

A) A photograph of ACG‐GelMA hydrogel scaffold printed by air‐extrusion method assisted with a low‐temperature receiver before UV light irradiation; B) microscope image of PACG‐GelMA hydrogel scaffold after UV light irradiation and reaching swelling equilibrium (stained with rhodamine); C) compressive stress–strain curves of the printed porous PACG10‐GelMA10‐Mn²⁺ hydrogel scaffolds, PACG35‐GelMA7‐BG hydrogel scaffolds, and gradient hydrogel scaffolds; D) cyclic compressive stress‐strain curves for the printed gradient scaffold (the top three layers were printed by PACG10‐GelMA10 hydrogel, and the bottom nine layers were printed by PACG35‐GelMA7‐BG hydrogel under a maximum strain of 30%. The cycle numbers were set as 100.

Figure 5

Gene analysis for chondrogenic differentiation and osteogenic differentiation from hBMSCs grown in different scaffolds. A,B) The expression of cartilage‐associated gene (COL II, aggrecan, SOX‐9, and COL I) after incubated for 7 and 14 days, respectively; C,D) the expression of osteogenesis‐associated gene (ALP, OCN, COL I, and RUNX2) after incubated for 7 and 14 days, respectively. Mn²⁺‐loaded PACG10‐GelMA10 hydrogel scaffold or BG‐loaded PACG35‐GelMA7 hydrogel scaffold significantly elevates the chondrogenic or osteogenic differentiation of hBMSCs, respectively (*p < 0.05, ^# p < 0.01, compared with control groups).

Figure 6

Characteristic 3D reconstruction images of micro‐CT analysis of the repaired subchondral bone at 4, 8, and 12 weeks in different groups.

Figure 7

Histological assessment of repaired cartilage subchondral bone at 8 and 12 weeks postsurgery in different groups. A) Hematoxylin and eosin (H&E), toluidine blue (T‐B) staining, and immunohistological staining for Coll II, GAGs, COL I, and OCN, show simultaneously enhanced cartilage and subchondral bone repair in the PAG‐Mn‐BG group, compared to the PAG group and the untreated control (blank) (N: normal cartilage; R: repair cartilage; the arrows indicate the margins of the normal cartilage and repaired cartilage; Scale bar = 200 µm); B) compression destruction tests of the repaired knees at 12 weeks postsurgery in different groups (^# p < 0.01, compared with the normal knees); C,D) quantification analysis of interleukin‐1 (1L‐1β) and tumor necrosis factor‐α (TNF‐α) in the serum of experimental rats at 1, 2, 3, 4, 8, and 12 weeks after surgery (^# p < 0.01, compared with 1 week at the same group).