Particulate ECM biomaterial ink is 3D printed and naturally crosslinked to form structurally-layered and lubricated cartilage tissue mimics

Abstract

Articular cartilage is a layered tissue with a complex, heterogenous structure and lubricated surface which is challenging to reproduce using traditional tissue engineering methods. 3D printing techniques have enabled engineering of complex scaffolds for cartilage regeneration, but constructs fail to replicate the unique zonal layers, and limited cytocompatible crosslinkers exist. To address the need for mechanically robust, layered scaffolds, we developed an extracellular matrix particle-based biomaterial ink (pECM biomaterial ink) which can be extruded, polymerizes via disulfide bonding, and restores surface lubrication. Our cartilage pECM biomaterial ink utilizes functionalized hyaluronan, a naturally occurring glycosaminoglycan, crosslinked directly to decellularized tissue particles (ø 40-100 µm). We experimentally determined that hyaluronan functionalized with thiol groups (t-HA) forms disulfide bonds with the ECM particles to form a 3D network. We show that two inks can be co-printed to create a layered cartilage scaffold with bulk compressive and surface (friction coefficient, adhesion, and roughness) mechanics approaching values measured on native cartilage. We demonstrate that our printing process enables the addition of macropores throughout the construct, increasing the viability of introduced cells by 10%. The delivery of these 3D printed scaffolds to a defect is straightforward, customizable to any shape, and adheres to surrounding tissue.

Preparation and Use of Decellularized Extracellular Matrix for Tissue Engineering

“…The first step of solubilizing dECM is to turn it into a powder. There are various methods for doing this, such as freezing with liquid nitrogen and using a mortar and pestle [ 82 , 91 , 125 , 136 , 159 , 161 , 212 ], milling with a rotary or cryogenic mill [ 20 , 23 , 87 , 110 , 138 , 139 , 168 , 171 , 175 , 201 , 223 , 237 , 279 , 320 , 321 ], or making a slurry with a homogonizer, followed by lyophilizing [ 302 ]. A variation on this process involves saturating the dECM with NaCl, snap freezing the dECM to precipitate the NaCl crystals and separate the protein fibres, and then milling it into a powder, followed by multiple washes to remove the NaCl, centrifugation, snap freezing of the pellet, and lyophilizing and disaggregating the powder; however, this process is not often used for preparing dECM for solubilizing [ 321 , 322 ].…”

“…A variation on this process involves saturating the dECM with NaCl, snap freezing the dECM to precipitate the NaCl crystals and separate the protein fibres, and then milling it into a powder, followed by multiple washes to remove the NaCl, centrifugation, snap freezing of the pellet, and lyophilizing and disaggregating the powder; however, this process is not often used for preparing dECM for solubilizing [ 321 , 322 ]. Once the dECM has turned into a powder of suitable particle size, the powder is lyophilized (if not done before milling), and then it can either be used directly as an additive [ 102 , 175 , 279 , 280 , 323 ] or be digested to create a liquid dECM mixture.…”

3D-biofabricated chondrocyte-laden decellularized extracellular matrix-contained gelatin methacrylate auxetic scaffolds under cyclic tensile stimulation for cartilage regeneration

“…The pore size of D0, D0.5, and D1 is 524 ± 73 nm, 913 ± 102 nm, and 1438 ± 317 nm, respectively. In addition, the porosity means the potential of adhesion areas and migration space for cells in the hydrogel-based scaffold [39]. According to figure 3(B), there was no significant difference (p > 0.05) in porosity between groups as the matrix concentration increased and all were close to 86%.…”

Designing functional hyaluronic acid-based hydrogels for cartilage tissue engineering

“…fabricated a layered 3D bioprinted hydrogel utilizing HA-SH and ECM particles through disulfide bonds, which exhibited customizable specific shapes and mechanical properties similar to those of the native cartilage. Moreover, the hydrogel enhanced the viability of encapsulated chondrocytes [ 173 ].…”