Cyclic freeze–thaw grinding to decellularize meniscus for fabricating porous, elastic scaffolds

Abstract

Decellularized meniscus extracellular matrix (dmECM)‐based biological scaffolds in the forms of sponge, hydrogel, nanofiber, and composite have gained increasing interest in meniscus tissue engineering and regeneration. A common shortcoming of those scaffolds is insufficient mechanical strength and poor elasticity. Herein, we report a practicable protocol for milder meniscus decellularization to prepare elastic, porous dmECM scaffolds. Porcine meniscus was pulverized by cyclic freeze–thaw grinding and then treated with DNase to obtain fine dmECM particles. Individual dmECM particles were condensed to bulk preparation by centrifuge, followed by lyophilization to form blocks, and finally crosslinked by dehydrothermal treatment to obtain porous dmECM scaffolds. Our results show that the freeze–thaw grinding method was effective in removing cellular DNA with good retention of meniscus‐derived bioactive components. The dmECM scaffold had porous structure with interconnected mesopores and good mechanical properties. Primary articular chondrocytes proliferated robustly and maintained chondrogenic characteristics and produce abundant collagen on dmECM scaffolds. Evaluation of biocompatibility in a rat model shows that the dmECM scaffold elicited minor foreign body reactions, indicating effective antigen removal from dmECM. This study provides an alternative for preparing dmECM and fabricating porous scaffolds for meniscus repair and regeneration.

Applications and prospects of different functional hydrogels in meniscus repair

“…In order to further detect the regionally biochemical differences of hydrogels from different regions, Wu et al fabricated regionally decellularized meniscal ECM hydrogels with different (outer, middle, and inner) zones of porcine meniscus, and found that the cell-seeded outer meniscus (OM) hydrogel had a nine-fold increase in peak compressive strengths (18.3 ± 3.6 vs. 2.1 ± 0.1 kPa) and a six-fold increase in initial modulus (9.9 ± 2.6 vs. 1.7 ± 0.2 kPa), the cell-seeded middle meniscus (MM) hydrogel experienced a 21-fold increase in peak compressive strengths (27.1 ± 4.6 vs. 1.3 ± 0.1 kPa) and a nine-fold increase in initial modulus (6.7 ± 1.6 vs. 0.8 ± 0.1 kPa), the cell-seeded inner meniscus (IM) hydrogel achieved a 22-fold increase in peak compressive strengths (26.0 ± 3.0 vs. 1.2 ± 0.2 kPa) and a nine-fold increase in initial modulus (9.4 ± 1.8 vs. 0.9 ± 0.2 kPa) over the IM hydrogel only ( Wu et al, 2021 ). Ding et al compared the scaffold properties of native meniscus, untreated extracellular matris (ECM) and decellularized ECM (dmECM) from porcine meniscus, and found that both dmECM and untreated ECM scaffolds had lower compressive modulus than native meniscus (181 ± 63 kPa, p < 0.001) and the native meniscus was relatively very stiff and showed significantly higher failure compression stresses (2030 ± 250 kPa, p < 0.001) ( Ding Y. et al, 2022 ). From these two studies, it can be seen that although biological hydrogels can improve the mechanical strength by adding cells, they are still difficult to achieve the mechanical strength of normal meniscus.…”

A comparative study on various cell sources for constructing tissue-engineered meniscus

“…Cartilage tissues were then digested in 0.2% collagenase type II for 8–10 h at 37°C and filtrated through a 70 μm cell strainer. Chondrocytes were expanded in high glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin for two passages before cell seeding ( Ding Y et al, 2022 ).…”

Elastic 3D-Printed Nanofibers Composite Scaffold for Bone Tissue Engineering

“…The porosity of SA, SSP, and SSF scaffolds was determined by the liquid displacement method with pouring cylindrical-shaped scaffolds. 16 Briefly, the diameters (d), heights (h), and weights (m a ) of the dry scaffolds were measured, and the scaffolds were then immersed in 200-proof ethanol. The scaffolds were gently wiped with a paper towel and weighed (m b ) after removal from the ethanol.…”