Dan Wei

Photo-cross-linkable methacrylated gelatin and hydroxyapatite hybrid hydrogel for modularly engineering biomimetic osteon.

Modular tissue engineering holds great potential in regenerating natural complex tissues by engineering three-dimensional modular scaffolds with predefined geometry and biological characters. In modular tissue-like construction, a scaffold with an appropriate mechanical rigidity for assembling fabrication and high biocompatibility for cell survival is the key to the successful bioconstruction. In this work, a series of composite hydrogels (GH0, GH1, GH2, and GH3) based on a combination of methacrylated gelatin (GelMA) and hydroxyapatite (HA) was exploited to enhance hydrogel mechanical rigidity and promote cell functional expression for osteon biofabrication. These composite hydrogels presented a lower swelling ratio, higher mechanical moduli, and better biocompatibility when compared to the pure GelMA hydrogel. Furthermore, on the basis of the composite hydrogel and photolithograph technology, we successfully constructed an osteon-like concentric double-ring structure in which the inner ring encapsulating human umbilical vascular endothelial cells (HUVECs) was designed to imitate blood vessel tubule while the outer ring encapsulating human osteoblast-like cells (MG63s) acts as part of bone. During the coculture period, MG63s and HUVECs exhibited not only satisfying growth status but also the enhanced genic expression of osteogenesis-related and angiogenesis-related differentiations. These results demonstrate this GelMA-HA composite hydrogel system is promising for modular tissue engineering.

A spatial patternable macroporous hydrogel with cell-affinity domains to enhance cell spreading and differentiation

Cell adhesion and spreading are two essential factors for anchorage-dependent cells such as osteocytes. An adhesive macroporous hydrogel system, in which cell-affinitive domains and sufficient cytoskeleton reorganization space were simultaneously constructed, was proposed in this report to support cell adhesion and spreading, respectively, and facilitate cell differentiation and function establishment eventually. The adhesive macroporous alginate hydrogel was developed by RGD peptide graft and gelatin microspheres hybridization to generate cellular adhesion sites and highly interconnected macropores. The successful stretched morphology and enhanced osteogenic differentiation of MG-63 cells in this modified alginate hydrogel showed clearly the feasibility that cell function may be effectively facilitated. Besides, this hydrogel model can be further applied to construct complex micropatterned structure, such as individual microgels in shapes of circle, square, cross and ring, and osteon-like structure containing both osteogenic and vascularized area generated by a double-ring assembly. These results should provide this adhesive macroporous photocrosslinkable hydrogel system as potential three-dimensional scaffolds for guiding tissue formation, especially for the bioengineering of tissues that have multiple cell types and require precisely defined cell-cell and cell-substrate interactions.

Ultrasonication and Genipin Cross-Linking to Prepare Novel Silk Fibroin–Gelatin Composite Hydrogel

Hydrogels are highly desirable tissue engineering scaffolds due to their high water content and structural similarity to a natural extra cellular matrix. However, the extensive use of hydrogels is limited by their low strength and facile degradation. By combining mechanical integrity and slow degradation of silk fibroin with excellent bioactivity of gelatin, a novel biocompatible protein-based composite hydrogel of silk fibroin and gelatin was developed. The gelation of silk fibroin aqueous solution was accelerated by ultrasonication, and gelatin derived from porcine skin was immobilized into the hydrogel network by the silk fibroin β-sheets. After that, genipin was used to post-cross-link the hydrogel to form a compact and stable hydrogel network. This hydrogel composite was a mechanically robust biomaterial with predictable long-term degradation characteristics. MG63 cells readily attached, spread, and proliferated on the surface of the hydrogels as demonstrated by fluorescein diacetate/propidium iodide staining and mitochondrial activity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Furthermore, the physicochemical and biological properties of hydrogel were fine-tunable by altering the ratio of silk fibroin and gelatin. The silk fibroin/gelatin composite hydrogels are anticipated to have the potential as cartilage or non-load-bearing bone tissue engineering and regeneration.

Microfluidic-based generation of functional microfibers for biomimetic complex tissue construction

UNLABELLED Microfluidic-based fiber system displays great potential in reconstructing naturally complex tissues. In these systems, fabrication of the basic fiber is a significant factor in ensuring a functional construction. The fiber should possess the strong mechanical rigidity for assembly, predefined microenvironment for cell spatial distribution and high biocompatibility for cell functional expression. Herein we presented a composite material by the combination of methacrylated gelatin (GelMA) and alginate for fiber engineering with capillary microfluidic device. Being regulated by GelMA incorporation, the composite hydrogels exhibited higher mechanical moduli, better stretching performance, and lower swelling compared to pure alginate one. On the basis of the composite material and capillary microfluidic device, we constructed the double-layer hollow microfibers to simulate complex tissues. The microfibers could be precisely controlled in size and multi-layered structure by varying flow rates and outlet diameter, and it showed satisfied application in woven-structure assembly. As an example to mimic a functional tissue, a biomimetic osteon-like structure was fabricated by encapsulating human umbilical vascular endothelial cells (HUVECs) in middle layer to imitate vascular vessel and human osteoblast-like cells (MG63) in the outer layer to act role of bone. During the incubation period, both MG63 and HUVECs exhibited not only a robust growth, but also up-regulated gene expression. These results demonstrated this microfluidic-based composite microfibers system is a promising alternative in complex tissue regeneration. STATEMENT OF SIGNIFICANCE Cell-laden microfibers based on microfluidic device is attracting interest for reconstructing naturally complex tissues. One shortage is the lack of suitable materials which satisfy microfluidic fabrication and cell biofunctional survival. This study reports the first combination of alginate-GelMA composite and capillary-based microfluidic technology. The composite materials possess high mechanical properties for fabrication and assembly, and tunable environment for cell spatial encapsulation. Significantly, the engineered double-layer hollow microfiber with osteon-like structure showed enhanced cellular bioactivity and realized initially functional establishment. This microfluidic-based composite microfiber not only explores a competitive candidate in complex tissues reconstruction, but also expands the biological application of microfluidic technology. This developing interdisciplinary area should be widely interested to the readers of biofabrication, biomaterials and tissue engineering.

Continuous Fabrication and Assembly of Spatial Cell-Laden Fibers for a Tissue-Like Construct via a Photolithographic-Based Microfluidic Chip

Engineering three-dimensional (3D) scaffolds with in vivo like architecture and function has shown great potential for tissue regeneration. Here we developed a facile microfluidic-based strategy for the continuous fabrication of cell-laden microfibers with hierarchically organized architecture. We show that photolithographically fabricated microfluidic devices offer a simple and reliable way to create anatomically inspired complex structures. Furthermore, the use of photo-cross-linkable methacrylated alginate allows modulation of both the mechanical properties and biological activity of the hydrogels for targeted applications. Via this approach, multilayered hollow microfibers were continuously fabricated, which can be easily assembled in situ, using 3D printing, into a larger, tissue-like construct. Importantly, this biomimetic approach promoted the development of phenotypical functions of the target tissue. As a model to engineer a complex tissue construct, osteon-like fiber was biomimetically engineered, and enhanced vasculogenic and osteogenic expression were observed in the encapsulated human umbilical cord vein endothelial cells and osteoblast-like MG63 cells respectively within the osteon fibers. The capability of this approach to create functional building blocks will be advantageous for bottom-up regeneration of complex, large tissue defects and, more broadly, will benefit a variety of applications in tissue engineering and biomedical research.

Photo-crosslinked mono-component type II collagen hydrogel as a matrix to induce chondrogenic differentiation of bone marrow mesenchymal stem cells

Type II collagen is a prospective chondro-inductive matrix for bone marrow mesenchymal stem cells (BMSCs), a key component of the extracellular matrix of cartilage; however, its application is limited by deficient fibrillogenesis and gelation. Herein, type II collagen methacrylamide (Col-II-MA) was synthesized by an amidation reaction between the ε-amino groups on collagen lysine and methacrylic anhydride to enable photo-crosslinking of the collagen, thus accomplishing a one-step preparation of mono-component type II collagen hydrogel for the first time. BMSCs encapsulated within the Col-II-MA hydrogel exhibited accelerated proliferation and morphological changes that are similar to chondrogenesis, as well as up-regulated expression of chondrogenic genes and remarkable secretion of the cartilaginous matrix. These results demonstrated that this effective synthetic approach facilitated the formation of photo-active type II collagen hydrogel with a well-preserved triple helical conformation, which provides BMSCs with a favorable microenvironment for growth and the essential chondro-inductive matrix for differentiation. Furthermore, the hydrogel is applicable to microfabrication techniques and displays promise for future applications in microscale tissue engineering.

The development of cell-initiated degradable hydrogel based on methacrylated alginate applicable to multiple microfabrication technologies

Biomimetic multicellular complex tissues can be obtained via 3D microfabrication approaches, although the lack of active materials that meet the needs of both molding and the maintenance of cell function is the main bottleneck in their application. The development of 3D cell culture platforms, which are suitable for multiple cell types and various microfabrication technologies, would open the way to the biomimetic construction of multicellular complex tissues. In this study, a MMP-sensitive photocrosslinkable hydrogel system based on methacrylated alginate, which is suitable for the survival of multiple cell types and can be employed in a variety of micro-manufacturing technologies, was designed for the construction of 3D microfeatures to meet both the demands of molding and the requirement for biomimetic function. By employing the characteristics of photocrosslinking and ion crosslinking, the gel system was combined with a variety of microfabrication techniques to construct biomimetic microstructures with a spatial distribution of multiple types of cell. The diversity of shapes and cell types in these assemblies, as well as the maintenance of cell activity, collectively show that the functional MMP-sensitive gel system has the potential to be used as a cell culture platform for the construction of tissue microstructures.

Methacrylamide-modified Collagen Hydrogel with Improved Anti-actin-mediated Matrix Contraction Behavior

For an ideal biomimetic microenvironment to realize reliable cartilage regeneration, the ability to induce mesenchymal stem cell (MSCs) differentiation along the chondrogenic lineage and prevent further dedifferentiation is expected. With native bioactivity, collagen has been proved to be preferential for inducing the chondrogenic differentiation of MSCs. However, the phenotypic maintenance of differentiated chondrocytes in a collagen matrix is still a challenge. Actin traction, which causes drastic contraction of the collagen matrix, is frequently observed and might be an important factor that affects cell fates including chondrogenic differentiation and phenotypic maintenance. In this study, photochemical modification was applied to acquire collagen hydrogels with improved mechanical strength and creep behavior. Accompanied by inherited bioactivity, the photo-crosslinked collagen hydrogel well supported the actin cytoskeleton functionalization while resisting the actin-mediated matrix contraction. Benefitting from this, the hydrogel system promoted MSCs proliferation and chondrogenic differentiation, and more importantly, prevented further dedifferentiation. By exploring the mesenchymal development-related signal transduction markers, it was revealed that the promoted chondrogenesis was achieved through inhibiting the over-expression of MAPK and Wnt/β-catenin signaling pathways that up-regulated dedifferentiated gene expression. The strategy of applying the hydrogel system to cartilage regeneration is foreseeable based on the positive heterotopic and orthotopic chondrogenic differentiation.

Wet-spinning fabrication of shear-patterned alginate hydrogel microfibers and the guidance of cell alignment

Abstract Native tissue is naturally comprised of highly-ordered cell-matrix assemblies in a multi-hierarchical way, and the nano/submicron alignment of fibrous matrix is found to be significant in supporting cellular functionalization. In this study, a self-designed wet-spinning device appended with a rotary receiving pool was used to continuously produce shear-patterned hydrogel microfibers with aligned submicron topography. The process that the flow-induced shear force reshapes the surface of hydrogel fiber into aligned submicron topography was systematically analysed. Afterwards, the effect of fiber topography on cellular longitudinal spread and elongation was investigated by culturing rat neuron-like PC12 cells and human osteosarcoma MG63 cells with the spun hydrogel microfibers, respectively. The results suggested that the stronger shear flow force would lead to more distinct aligned submicron topography on fiber surface, which could induce cell orientation along with fiber axis and therefore form the cell-matrix dual-alignment. Finally, a multi-hierarchical tissue-like structure constructed by dual-oriented cell-matrix assemblies was fabricated based on this wet-spinning method. This work is believed to be a potentially novel biofabrication scheme for bottom-up constructing of engineered linear tissue, such as nerve bundle, cortical bone, muscle and hepatic cord.

Automated fabrication of hydrogel microfibers with tunable diameters for controlled cell alignment

A newly designed spinning device was utilized to produce continuous hydrogel microfibers with tunable diameters. It was found that the diameter of the microfiber was dependent on perfusion speed and coagulation wheel rotation rate. Their correlation was finally described by a mathematical expression, which proved to be useful for a size-tunable spinning technique. Based on the controllable fabrication of hydrogel microfibers with desired size, microfiber/cell-size-dependent cellular orientated spreading was studied by using PC12 and L929 as model cells. By further demonstrating the assembly of fibrous tissue-like grafts using the spun hydrogel microfibers, the wet spinning protocol was proved to be instructive for manufacturing size-tunable hydrogel microfibers, as well as two-dimensional or three-dimensional scaffolds with varied micro-structure for tissue engineering.