TEMPO-Oxidized Cellulose Nanofiber-Alginate Hydrogel as a Bioink for Human Meniscus Tissue Engineering

Abstract

Objective: The avascular inner regions of the knee menisci cannot self-heal. As a prospective treatment, functional replacements can be generated by cell-based 3D bioprinting with an appropriate cell source and biomaterial. To that end, human meniscus fibrochondrocytes (hMFC) from surgical castoffs of partial meniscectomies as well as cellulose nanofiber-alginate based hydrogels have emerged as a promising cell source and biomaterial combination. The objectives of the study were to first find the optimal formulations of TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl)-oxidized cellulose nanofiber/alginate (TCNF/ALG) precursors for bioprinting, and then to use them to investigate redifferentiation and synthesis of functional inner meniscus-like extracellular matrix (ECM) components by expanded hMFCs.Methods: The rheological properties including shear viscosity, thixotropic behavior recovery, and loss tangent of selected TCNF/ALG precursors were measured to find the optimum formulations for 3D bioprinting. hMFCs were mixed with TCNF/ALG precursors with suitable formulations and 3D bioprinted into cylindrical disc constructs and crosslinked with CaCl2 after printing. The bioprinted constructs then underwent 6 weeks of in vitro chondrogenesis in hypoxia prior to analysis with biomechanical, biochemical, molecular, and histological assays. hMFCs mixed with a collagen I gel were used as a control.Results: The TCNF/ALG and collagen-based constructs had similar compression moduli. The expression of COL2A1 was significantly higher in TCNF/ALG. The TCNF/ALG constructs showed more of an inner meniscus-like phenotype while the collagen I-based construct was consistent with a more outer meniscus-like phenotype. The expression of COL10A1 and MMP13 were lower in the TCNF/ALG constructs. In addition, the immunofluorescence of human type I and II collagens were evident in the TCNF/ALG, while the bovine type I collagen constructs lacked type II collagen deposition but did contain newly synthesized human type I collagen.

Applications and prospects of different functional hydrogels in meniscus repair

“…Frontiers in Bioengineering and Biotechnology frontiersin.org The TCNF/ALG constructs showed more of an inner meniscus-like phenotype while the collagen I-based construct was consistent with a more outer meniscus-like phenotype Lan et al (2021) (Continued on following page)…”

“…Lan et al fabricated TEMPO (2,2,6,6tetramethylpiperidine-1-oxyl)-oxidized cellulose nanofiber/ alginate (TCNF/ALG) hydrogel and suggested that human meniscus fibrochondrocytes (hMFC) implanted in the hydrogel exhibited a more inner meniscus-like phenotype, whereas cells cultured in a collagen I-based construct exhibited a more outer meniscus-like phenotype (Lan et al, 2021). As another common hydrogel ingredient, agarose is widely used to prepare gels for separating macromolecular A 3D printing wedge-shaped poly (ε-caprolactone) (PCL) scaffold as a backbone, in vitro cell study and scaffolds were implanted in the meniscus defect and sutured to the residual rim after the mature New Zealand White rabbits underwent total medial meniscectomy except 5% of the external rim in vivo 2% of meniscus extracellular matrix (MECM)-based hydrogel strongly enhanced chondrogenic marker mRNA expression and cell proliferation; PCL-hydrogel-MFCs group exhibited markedly better gross appearance and cartilage protection than other groups and the regenerated menisci in the PCL-hydrogel-MFCs group had similar histological structures, biochemical contents and biomechanical properties as the native menisci in the sham operation group Chen et al (2019) Porcine meniscal fibrochondrocytes Agorose, methacrylated gelatin (GelMA), methacrylated hyaluronic acid (MeHA) and GelMA-MeHA blend hydrogels Fibrochondrocytes (passage 2) were reconstituted in the polymer solutions prior to photocuring and then cultured in fibrochondrogenic medium Hydrogels have a higher potential for meniscal regeneration than the 3D printed PCL, and fibrochondrocytes could be directed to proliferate or produce cartilaginous or fibrocartilaginous ECM.…”

“…Meniscal fibrochondrocytes are the main cellular components of the meniscus. Multiple studies have shown that meniscal fibrochondrocytes can form meniscus-like tissue in vitro and promote meniscus regeneration in vivo ( Simson et al, 2013 ; Chen et al, 2019 ; Lan et al, 2021 ). Simson et al encapsulated bovine meniscal fibrochondrocytes in chondroitin sulfate (CS)-bone marrow (BM) hydrogel (CSBM) hydrogels, and found that meniscal fibrochondrocytes were able to survive, proliferate, and produce meniscus ECM in vitro , and meniscus explants adhered by C30B70 fused together after 12 weeks’ implantation in a subcutaneous model of athymic rats ( Simson et al, 2013 ).…”

A Guide to Polysaccharide-Based Hydrogel Bioinks for 3D Bioprinting Applications

“…The high diversity of chemical structures and properties of these plant polysaccharides opens the possibility for their application in several fields, including in the food area (e.g., as gelling agents) [ 97 ], in the biomedical field (e.g., in drug delivery systems) [ 98 ], and, more recently, in tissue engineering [ 99 ]. The interest in using some of these plant-derived polysaccharides in the formulation of bioinks for 3D bioprinting applications has also grown considerably in the later years, as summed up in Table 2 , with several works using cellulose and nanocelluloses [ 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 , 114 , 115 , 116 , 117 , 118 ] and pectin [ 119 , 120 ].…”

“…CELLINK Bioink from Cellink [ 132 ]; throughout the years, not much evolution and creativity has been observed regarding the use of NFC as a bioink component. Most recent studies still describe its combination with alginate for bioprinting of different cell lines, namely human cartilage [ 115 , 116 ], human dermis [ 117 ], bone [ 102 , 118 ], and skeletal muscle [ 103 ]. Additionally, nanocelluloses are still mainly used as reinforcing components for hydrogel bioinks due to their excellent mechanical properties.…”