Femtosecond Laser Maskless Optical Projection Lithography of Cartilage PCM Inspired 3D Protein Matrix to Chondrocyte Phenotype.

Hydrogels containing chondrocytes have exhibited excellent potential in regenerating hyaline cartilage. However, chondrocytes are vulnerable to dedifferentiation during in vitro culture, leading to fibrosis and mechanical degradation of newly formed cartilage. It is proposed to modulate cartilage formation via the developed chondrocyte pericellular matrix (PCM) -like scaffolds for the first time, in which the S, M, and L-sized scaffolds are fabricated by femtosecond laser maskless optical projection lithography (FL-MOPL) of bovine serum albumin-glyceryl methacrylate hydrogel. Chondrocytes on the M PCM-like scaffold can maintain round morphology and synthesize extracellular matrix (ECM) to induce regeneration of hyaline cartilage microtissues by geometrical restriction. A series of M PCM-like scaffolds is fabricated with different stiffness and those with a high Young's modulus are more effective in maintaining the chondrocyte phenotype. The proposed PCM-like scaffolds are effective in modulating cartilage formation influenced by pore size, depth, and stiffness, which will pave the way for a better understanding of the geometric cues of mechanotransduction interactions in regulating cell fate and open up new avenues for tissue engineering.

Multipatterned Chondrocytes' Scaffolds by FL-MOPL with a BSA-GMA Hydrogel to Regulate Chondrocytes' Morphology.

Repairing articular cartilage damage is challenging due to its low regenerative capacity. In vitro, cartilage regeneration is a potential strategy for the functional reconstruction of cartilage defects. A hydrogel is an advanced material for mimicking the extracellular matrix (ECM) due to its hydrophilicity and biocompatibility, which is known as an ideal scaffold for cartilage regeneration. However, chondrocyte culture in vitro tends to dedifferentiate, leading to fibrosis and reduced mechanical properties of the newly formed cartilage tissue. Therefore, it is necessary to understand the mechanism of modulating the chondrocytes' morphology. In this study, we synthesize photo-cross-linkable bovine serum albumin-glycidyl methacrylate (BSA-GMA) with 65% methacrylation. The scaffolds are found to be suitable for chondrocyte growth, which are fabricated by homemade femtosecond laser maskless optical projection lithography (FL-MOPL). The large-area chondrocyte scaffolds have holes with interior angles of triangle (T), quadrilateral (Q), pentagon (P), hexagonal (H), and round (R). The FL-MOPL polymerization mechanism, swelling, degradation, and biocompatibility of the BSA-GMA hydrogel have been investigated. Furthermore, cytoskeleton and nucleus staining reveals that the R-scaffold with larger interior angle is more effective in maintaining chondrocyte morphology and preventing dedifferentiation. The scaffold's ability to maintain the chondrocytes' morphology improves as its shape matches that of the chondrocytes. These results suggest that the BSA-GMA scaffold is a suitable candidate for preventing chondrocyte differentiation and supporting cartilage tissue repair and regeneration. The proposed method for chondrocyte in vitro culture by developing biocompatible materials and flexible fabrication techniques would broaden the potential application of chondrocyte transplants as a viable treatment for cartilage-related diseases.

Synthesis of biocompatible BSA-GMA and two-photon polymerization of 3D hydrogels with free radical type I photoinitiator.

Although the development of three-dimensional (3D) printing technology is growing rapidly in the biomedical field, it remains a challenge to achieve arbitrary 3D structures with high resolution and high efficiency. Protein hydrogels fabricated by two- photon polymerization (TPP) have excellent mechanical properties, high precision, and 3D architecture. However, a large number of the amino acid group in bovine serum albumin (BSA) would be consumed when the protein-based hydrogels use dyes of free radical type II photoinitiators. In this study, we use glycidyl methacrylate (GMA) to modify BSA molecules to obtain a series of BSA-GMA materials, allowing the protein material to be two-photon polymerized with a water-soluble free radical type I photoinitiator. The precisely controllable 3D structure of the BSA-GMA hydrogel was fabricated by adjusting the concentration of the precursor solution, the degree of methacrylation, and the processing parameters of the TPP technique. Importantly, BSA-GMA materials are free of acidic hazardous substances. Meanwhile, the water-soluble initiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphite (LAP) allows TPP on the vinyl group of the GMA chain and thus without consuming its amino acid group. The as-prepared BSA-GMA hydrogel structure exhibits excellent autofluorescence imaging, pH responsiveness, and biocompatibility, which would provide new avenues for potential applications in tissue engineering and biomedical fields to meet specific biological requirements.

22 nm Resolution Achieved by Femtosecond Laser Two-Photon Polymerization of a Hyaluronic Acid Vinyl Ester Hydrogel.

Three-dimensional (3D) bioinspired hydrogels have played an important role in tissue engineering, owing to their advantage of excellent biocompatibility. Here, the two-photon polymerization (TPP) of a 3D hydrogel with high precision has been investigated, using the precursor with hyaluronic acid vinyl ester (HAVE) as the biocompatibility hydrogel monomer, 3,3'-((((1E,1'E)-(2-oxocyclopentane-1,3-diylidene) bis(methanylylidene)) bis(4,1-phenylene)) bis(methylazanediyl))dipropanoate as the water-soluble initiator, and dl-dithiothreitol (DTT) as the click-chemistry cross-linker. The TPP properties of the HAVE precursors have been comprehensively investigated by adjusting the solubility and the formulation of the photoresist. The feature line width of 22 nm has been obtained at a processing laser threshold of 3.67 mW, and the 3D hydrogel scaffold structures have been fabricated. Furthermore, the average value of Young's modulus is 94 kPa for the 3D hydrogel, and cell biocompatibility has been demonstrated. This study would provide high potential for achieving a 3D hydrogel scaffold with highly precise configuration in tissue engineering and biomedicine.