One-Step Photoactivation of a Dual-Functionalized Bioink as Cell Carrier and Cartilage-Binding Glue for Chondral Regeneration

Abstract

Cartilage defects can result in pain, disability, and osteoarthritis. Hydrogels providing a chondroregeneration-permissive environment are often mechanically weak and display poor lateral integration into the surrounding cartilage. This study develops a visible-light responsive gelatin ink with enhanced interactions with the native tissue, and potential for intraoperative bioprinting. A dual-functionalized tyramine and methacryloyl gelatin (GelMA-Tyr) is synthesized. Photo-crosslinking of both groups is triggered in a single photoexposure by cell-compatible visible light in presence of tris(2,2'-bipyridyl)dichlororuthenium(II) and sodium persulfate as initiators. Neo-cartilage formation from embedded chondroprogenitor cells is demonstrated in vitro, and the hydrogel is successfully applied as bioink for extrusion-printing. Visible light in situ crosslinking in cartilage defects results in no damage to the surrounding tissue, in contrast to the native chondrocyte death caused by UV light (365-400 nm range), commonly used in biofabrication. Tyramine-binding to proteins in native cartilage leads to a 15-fold increment in the adhesive strength of the bioglue compared to pristine GelMA. Enhanced adhesion is observed also when the ink is extruded as printable filaments into the defect. Visible-light reactive GelMA-Tyr bioinks can act as orthobiologic carriers for in situ cartilage repair, providing a permissive environment for chondrogenesis, and establishing safe lateral integration into chondral defects.

Keywords: biofabrication; bioglue; bioprinting; cartilage tissue engineering; tissue integration.

Figure 1. Functionalization process to obtain the dual responsive hydrogels.

A) Schematic representation of the synthesis of GelMA and GelMA-Tyr; representative ¹H-NMR spectra of gelatin, GelMA and GelMA-Tyr. B–G) ¹H-NMR spectra of the functionalized hydrogels, showing the presence of the characteristic peaks for acrylates (a,b) in the GelMA and GelMA-Tyr groups and for the added phenolic groups in the GelMA-Tyr polymers.

Figure 2. Fabrication and physicochemical and mechanical characterization of the one-step inducible, dual crosslinked hydrogels.

A) Schematic representation of the crosslinking process of the GelMA-Tyr macromer. B) Sol fraction values and C) swelling behavior of the hydrogels as observed via sol–gel analysis; D) compressive young’s modulus of the casted hydrogels. Incorporation of myoglobin into GelMA or GelMA-Tyr hydrogels: E) release profile of myoglobin from GelMA or GelMA-Tyr hydrogels over 3000 min; F) macroscopic images of GelMA and GelMA-Tyr hydrogels incorporated with myoglobin over 48 h.

Figure 3. Chondrogenic differentiation of ACPCs encapsulated within GelMA or GelMA-Tyr hydrogels.

A) Cell viability after 1 and 7 days in culture. B) GAG/DNA and C) Young’s modulus of ACPC-laden GelMA or GelMA-Tyr gels after 1 and 28 days in culture. D) Collagen type II, E) Collagen type I, and F) PRG4 gene expression of ACPC-laden GelMA or GelMA-Tyr gels after 1 and 28 days in culture. Histological stainings of ACPC-laden GelMA or GelMA-Tyr gels after 28 days, G,J) safranin-O, H,K) collagen type II, scale bar = 20 μm, and I,L) collagen type 1, scale bar = 40 μm.

Figure 4. Extrusion bioprinting of GelMA-Tyr bioinks.

A) Rheological profile of GelMA and GelMA-Tyr in response to shear rate. B) Cell viability of ACPCs bioprinted within GelMA and GelMA-Tyr constructs. Macroscopic images of extrusion bioprinted C) GelMA and D) GelMA-Tyr constructs, together with a representative image of the filament collapse assay, showing the ability of the printed struts to bridge gaps of 4 mm without noticeable deformation, and up to 16 mm while experiencing sagging. Scale bar = 1 mm.

Figure 5

A) Schematic of intraoperative administration of GelMA-Tyr to the chondral defect. B) Normalized amount of live cells and C–F) LIVE/DEAD images of cartilage biopsies irradiated with UV or visible light. Scale bar = 100 μm. G) Setup of the pushout assay to determine bond-strength. H) Bond-strength of GelMA or GelMA-Tyr administered to the cartilage biopsies as a solution or physically crosslinked gel. I) Cartilage biopsies adhered together using GelMA-Tyr.