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Review
. 2024 Feb 24;14(5):1982-2035.
doi: 10.7150/thno.89493. eCollection 2024.

Highly oriented hydrogels for tissue regeneration: design strategies, cellular mechanisms, and biomedical applications

Jiuping Wu  1   2 , Zhihe Yun  3 , Wenlong Song  4 , Tao Yu  3 , Wu Xue  3 , Qinyi Liu  3 , Xinzhi Sun  1
Affiliations
Review

Highly oriented hydrogels for tissue regeneration: design strategies, cellular mechanisms, and biomedical applications

Jiuping Wu et al. Theranostics. .

Abstract

Many human tissues exhibit a highly oriented architecture that confers them with distinct mechanical properties, enabling adaptation to diverse and challenging environments. Hydrogels, with their water-rich "soft and wet" structure, have emerged as promising biomimetic materials in tissue engineering for repairing and replacing damaged tissues and organs. Highly oriented hydrogels can especially emulate the structural orientation found in human tissue, exhibiting unique physiological functions and properties absent in traditional homogeneous isotropic hydrogels. The design and preparation of highly oriented hydrogels involve strategies like including hydrogels with highly oriented nanofillers, polymer-chain networks, void channels, and microfabricated structures. Understanding the specific mechanism of action of how these highly oriented hydrogels affect cell behavior and their biological applications for repairing highly oriented tissues such as the cornea, skin, skeletal muscle, tendon, ligament, cartilage, bone, blood vessels, heart, etc., requires further exploration and generalization. Therefore, this review aims to fill that gap by focusing on the design strategy of highly oriented hydrogels and their application in the field of tissue engineering. Furthermore, we provide a detailed discussion on the application of highly oriented hydrogels in various tissues and organs and the mechanisms through which highly oriented structures influence cell behavior.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Highly oriented tissues and organs in the human body (Figure was created with Biorender.com).
Figure 2
Figure 2
Classification of highly oriented hydrogels, which can be broadly summarized as highly oriented nanofillers hydrogels obtained using strategies such as magnetic/electric fields and mechanical force, highly oriented polymer-chain network hydrogels obtained by strategies such as mechanical force and ion diffusion, highly oriented void channels hydrogels prepared by strategies such as directed freezing/ice template, and highly oriented hydrogels with microfabricated structures using strategies such as 3D printing.
Figure 3
Figure 3
Fabrication of highly oriented hydrogels based on 0D nanoparticles (A), 1D nanotubes (B) and 2D nanosheets (C) in an external magnetic field. A). Schematic illustration of the formation of anisotropic hTSPC-nanocomposite hydrogel induced by the addition of paramagnetic iron oxide nanoparticles (IOPs) under exposure to magnetic field and cartoon of the three study groups. Adapted with permission from , copyright 2021, Royal Society of Chemistry. B) Schematic representation of 1D nanorods in hydrogel oriented under an external magnetic field. Adapted with permission from , copyright 2020, Elsevier. C) Schematic representation of a) the fabrication of PNIPAm-MoS2/Fe3O4 hydrogel and b) programming the orientation of MoS2/Fe3O4 under the magnetic field followed by NIR light-treatment of frozen hydrogels. Representative SEM images of PNIPAm-MoS2/Fe3O4 hydrogels under different conditions. Adapted with permission from , copyrights 2022, Wiley-VCH.
Figure 4
Figure 4
A-F) Macroscopic and microscopic images of MNP-, MNP+MNA-, and MNP+MNA+. G) Schematic diagram of hydrogels with oriented structures by combining 3D printing with MNPs induced by magnetic fields. Adapted with permission from , copyright 2021, Nature Publishing Group. H) Schematic illustration of injectable oriented MSNF/Gel nanofiber hydrogel scaffold for biomimicking of living constructs with macro- and micro-structures in vitro and aligned regenerated myofibers in vivo. Adapted with permission from , copyright 2022, Elsevier.
Figure 5
Figure 5
A) a) Synthesis of 0D microcapsule assembly. b) The fabrication process of orienting 0D microcapsule alignment by DEP manipulation. B) Random distribution of 0D microcapsule and subsequent orientation alignment within the hydrogel after DEP. Adapted with permission from , copyright 2021, Elsevier. C) Schematic representation of the fabrication process for CNT alignment within the GelMA hydrogel and phase contrast images of the CNT alignment over time. CNTs were aligned after 20 seconds. Adapted with permission from , copyright 2013, Wiley-VCH. D) Scheme of preparation of 2D PNIPAm/GO oriented hydrogel. Adapted with permission from , copyright 2018, American Chemical Society.
Figure 6
Figure 6
A) Schematic of prepared highly oriented, fiber-aligned hydrogels using shear forces during extrusion printing. Adapted with permission from , copyright 2021, IOP Publishing. B) (a,b)Schematic of shear-mediated extrusion of highly oriented hydrogel from the mixture of CNCs, GelMA, and LAP using a microfluidic printhead, and (c) photograph of the hydrogel sheet. Adapted with permission from , copyright 2021, Wiley-VCH. C) Confocal microscopy images of random aligned hydrogels and highly oriented hydrogels, and schematic of preparation of highly oriented PPy nanocomposite hydrogels after repeated stretching. D) They have good biocompatibility, induce targeted growth of cardiomyocytes and promote restoration of electrical conductivity. Adapted with permission from , copyright 2021, American Chemical Society. E) Schematic representation of the synthesis procedures of oriented void channels hydrogels with an ordered internal orientation structure and further applications in wearable flexible sensors and 3D sensor arrays. Adapted with permission from , copyright 2021, Wiley-VCH. F) Schematic of self-assembled oriented structures of CNC arrangement in the polymer network after UV curing with different standing times Adapted with permission from , copyright 2021, Elsevier.
Figure 7
Figure 7
A) Photographs of the highly oriented Alg/PAM hydrogels during the RsEC process; remodeling (pre-stretching) and subsequent solvent exchange followed by ionic crosslinking (RsEC). B) Illustrations of the changes in the molecular structure during the RsEC process: (i) as-prepared Ca2+-crosslinked Alg/PAM hydrogel, (ii) pre-stretched hydrogel, (iii) solvent-exchanged hydrogel, and (iv) Al3+ crosslinked hydrogel. Adapted with permission from , copyright 2022, American Chemical Society. C) Design of the strong, stiff adhesive highly oriented TN hydrogel. The final TN-RsC highly oriented hydrogel subjected to the RsC process was cross-linked by Al3+ ions and exhibited strong mechanical and adhesion properties. D) a) Schematic of the real ligament and anisotropic structure of the ligament, and b) photographs of TN-RsC hydrogel mimic the real ligament. Adapted with permission from , copyright 2022, Wiley-VCH. E) Graphical illustrations for the production of oriented and gradient chitosan hydrogel films with hierarchical structure via the self-made mold. Adapted with permission from , copyright 2022, Elsevier.
Figure 8
Figure 8
A) Chemical structure of the rigid polyanion, PBDT, and schematic for the formation of anisotropic hydrogel by unidirectional diffusion of Ca2+ ions into PBDT solution from two opposite lateral sides. The diffusion induced the alignment of PBDT perpendicular to the diffusion direction near the entrance yet parallel to the diffusion direction at the middle region where the two fluxes met. Adapted with permission from , copyright 2019, WILEY‐VCH. B) Schematic illustration and effect of pre-shear bioprinting of highly oriented hydrogel microfiber-enabled oriented growth of cells. Adapted with permission from , copyright 2021, Royal Society of Chemistry. C) Process to Form Tough Silk Nanofiber Hydrogels with highly oriented Architectures. D) Characterization of SF hydrogels with highly oriented structures. Adapted with permission from , copyright 2020, American Chemical Society.
Figure 9
Figure 9
A) Schematic illustration for the highly oriented void channels hydrogels fabrication by using an ice-template freeze casting process. B) SEM images of the highly oriented void channels hydrogels. Adapted with permission from , copyright 2021, Wiley‐VCH. C) Schematic drawing of the void channels formed by unidirectional diffusion of divalent metal cations (Me2+) into a solution of sodium alginate, and cross-sections of the void channels in hydrogels formed by different cations. (a-h). Scanning electron microscopy images for highly oriented void channels hydrogels. Adapted with permission from , copyright 2022, Elsevier.
Figure 10
Figure 10
A) Schematic illustration of the composition and synthesis mechanism of the cellulose nanofibers + hyaluronic acid methacrylate (CN+HAMA) highly oriented hydrogels. B) SEM images of CNs, hyaluronic acid (HA), HAMA, and CN+HAMA hydrogels with different concentration ratios. Adapted with permission from , copyright 2022, AccScience Publishing. C) Schematic illustration of the proposed strategy to fabricate high-resolution oriented biomimetic constructs. D) The effect of short magnetically-responsive microfibers (sMRFs) orientation and concentration over the alignment and morphology of the encapsulated hASCs, along with their orientation plots. E) CLM images of hASCs adhered to sMRFs and acquiring elongated morphologies in highly oriented hydrogels, and viability and orientation of the hASCs encapsulated within oriented hydrogel after i) day 1, ii) day 3, iii) day 10 and iv/v/vi) day 21 of cell culture. F) Cell viability after 21 days in 3D bioprinted hydrogels with 2.00 mg·mL-1 of i) randomly-oriented sMRFs and ii) aligned sMRFs. G) CLM images of hASCs cytoskeleton in GelMA, i) tile scan of fabricated construct, ii) straight line of the printed filaments, iii) curve of the printed filaments. Adapted with permission from , copyright 2022, Wiley‐VCH.
Figure 11
Figure 11
A) (a) Schematic illustration of the procedure for the fabrication of the microstructured highly oriented hydrogel substrate. Scanning electron microscopic image (b), picture (c) and microscopic image (d) of hydrogel. Adapted with permission from , copyright 2016, American Chemical Society. B) a) Schematic of Travelling wave generation of PIC wrinkles on the hydrogel surface. b) Fabrication of wrinkles on a hydrogel surface by electrophoretic. C) a) Experimental electrophoretic formation of aligned wrinkles under the lateral compression of gels. b) Time-course images of aligned wrinkle formation during electrophoresis. c) Trajectories of aligned wrinkle as a function of time. Adapted with permission from , copyright 2022, Wiley‐VCH. D) a) Schematic image of highly oriented hydrogels fabrication. b) Differences in mechanical properties between the outside and inside of hydrogels. c,d) Birefringence patterns and magnified POM images of an hydrogels sample. e,f) Cross-section and surface SEM images of hydrogels showing a long-range and highly oriented self-wrinkling configuration. Adapted with permission from , copyright 2019, Wiley‐VCH.
Figure 12
Figure 12
A) a) Schematic of a conventional flexible and striated skeletal muscle tissue to be emulated by the wood hydrogel. b) Images of a 7 cm long wood hydrogel sample being twisted 180°. c) Depiction of the wood hydrogel hydrogen bonding and covalent cross-linking between PAM chains. B) Optical (a-f) and SEM images (g-i) of the wood hydrogel and white wood. Adapted with permission from , copyright 2018, Wiley‐VCH. C) Schematic illustration of hydrogen bonding formed between white sugarcane and pHEMA chains. Adapted with permission from , copyright 2021, Wiley‐VCH. D) Schematics illustrating the all-wood hydrogel preparation (a-f). Adapted with permission from , copyright 2021, Elesevier.
Figure 13
Figure 13
A) Schematic illustration of FLight strategy for fabricating highly oriented microfilaments and their cell guidance properties. B) Bright-field image of FLight hydrogel construct, magnified image of microfilaments structuring of oriented hydrogel, distribution of the microfilament diameter, and the distribution of angles' difference between the orientation of microfilament and direction of projection. C) Fluorescence images of cell-laden hydrogel matrix. D) Cell guidance properties of microfilaments and maturation of highly aligned tissue-engineered constructs. Evidence of highly oriented fibroblasts, tenocytes, endothelial cells, and myotubes by immunofluorescence staining. Adapted with permission from , copyright 2022, Wiley‐VCH.
Figure 14
Figure 14
A) Schematic illustration of NFYs-NET hydrogel preparation. B) (a) Cardiomyocytes seeded and cultured on NFYs-NET hydrogels. (b) Relative cells viability percentages of cardiomyocytes on NFYs-NET hydrogels evaluated by live/dead assay. (c) The top views and 3D views of fluorescent images of cardiomyocytes on NFYs-NET hydrogels. C) (a-d) Fabrication process of 1-layer oriented hydrogels, and their gross image, optical image, and the 3D view of confocal image. (e, f) Scheme of cardiomyocytes seeded and cultured within hydrogels and the fluorescent image of cardiomyocytes. (g, h, k) The confocal images showed the cellular alignment and elongation within oriented hydrogels while (i, j, l) the random morphology of cells within GelMA hydrogel, and (m-p) the quantitative analysis of cellular orientation distribution. D) (a-c) Schematic of myocardium oriented structure, multilayers orientation of NFYs-NETs assemble, and 2-layer oriented hydrogels fabrication. (d, e) The gross image and 3D view of confocal image of 2-layer 3D scaffolds. (f-h) The scheme of culturing cells within oriented hydrogels and fluorescent images of cardiomyocytes with horizontal direction and vertical direction. (i-l) The top view and 3D view of confocal images of cells and the quantitative analysis of cellular orientation distribution. E) (a) CMs were cultured on the NFYs-NET layer, ECs were encapsulated within hydrogel shell. (b-d) The fluorescent images of ECs, CMs and their merge image. (e-g) The 3D view of confocal images of ECs and CMs within scaffolds, and the quantitative analysis of cellular orientation distribution. (h, i) The distribution of ECs in hydrogel and CMs on NFYs-NET. Adapted with permission from , copyright 2017, American Chemical Society.
Figure 15
Figure 15
A) (a) Schematic of preparation of highly oriented mPCL fibers. (b-d) Different fiber spacing of the printed fibrous networks. (e) Schematic of preparation of highly oriented hydrogels, and their stereomicroscopy images. Adapted with permission from , copyright 2017, IOP Publishing. B) (a) Schematic illustration of the bilayer oriented heterogeneous hydrogel (BH-CF/MMT hydrogel). (b) SEM images of the BH-CF/MMT hydrogel. (c) Schematic illustration of the binding interface of the BH-CF/MMT hydrogel. Adapted with permission from , copyright 2022, American Chemical Society. C) (a) Schematic illustrates two types of wood frames that can be cut from a natural tree trunk. (b) The process of delignification. (c) The fabrication process of the bio-inspired three-zone highly oriented composite hydrogel. D) (a, d) Schemes of softwood and hardwood structure. (b, e) Longitudinal direction cross-sectional and (c, f) radial direction cross-sectional SEM images of the softwood and hardwood with aligned channels. E) (a-c) Schemes and cell morphologies of actin filaments and nuclei on the surface of glass slide, HL and SL. (d) 2D-FFT curves of the fluorescent images cultured on different material surfaces. (e-g) BMSCs relative proliferation rate and the relative content of collagen I and collagen II of different zone hydrogels. Adapted with permission from , copyright 2020, Elsevier.
Figure 16
Figure 16
A) Processing principles of the highly oriented chitin-tannic acid hydrogel. B) Field emission SEM images of a,e) CHTA-0; b,f) CHTA-30; c,g) CHTA-60; and d,h) CHTA-90 hydrogels. C) a-c) Fluorescence microscopy images of BMSCs on isotropic CHTA/Bru-0, oriented CHTA/Bru-90 hydrogels and their migration. d, e) Angle distribution histograms and the number of the BMSCs cultured on isotropic CHTA/Bru-0 and anisotropic CHTA/Bru-90 hydrogels. Adapted with permission from , copyright 2022, Wiley‐VCH. D) The flowchart of the experimental process, and the mechanism diagram of DFO promoting angiogenesis. Adapted with permission from , copyright 2022, Elsevier. E) a) Schematic of the hierarchical structure of natural bone. b) Schematic illustration of the fabrication approach and the structure of the MWH. F) a-m) Structural and compositional characterization of highly oriented MWH. Adapted with permission from , copyright 2021, Wiley-VCH.
Figure 17
Figure 17
A) a-f) Schematic of biofabrication and stimulation of 3D oriented cell-laden hydrogel yarns using the wet-spinning technique. B). Collagen I expressed by hBM-MSCs encapsulated into different hydrogels. Adapted with permission from , copyright 2019, Wiley-VCH. C) Schematic of biofabrication of cellulose nanocrystals (CNC) and hydroxyapatite (HA) oriented and aligned under a magnetic field. D) a-f) The nuclei aspect ratio, directionality of cell distribution, and tendon-related ECM protein TNC secretion were assessed by confocal immunofluorescence. Adapted with permission from , copyright 2019, American Chemical Society. E) Schematic of biofabrication of highly oriented ADC hydrogel scaffolds and the ability to tenogenic differentiation. F) Cellular morphology and collagen formation of hBM-MSC-seeded ADCs of 20% static strain (SS) and 20% cyclic rest (CR) and their controls, free-floating (FF) and tethered (T). Adapted with permission from , copyright 2022, Elsevier.
Figure 18
Figure 18
A) Schematic illustration of the fabrication of aligned MSNF/Gel scaffold in situ under magnetic field, and the fluorescence images showed that MSNF was oriented encapsulated within GelMA hydrogels. B) (a-c) Cytoskeleton morphology of C2C12 cells encapsulated within Gel, random MSNF/Gel scaffold and oriented MSNF/Gel scaffold after culturing for 3 days, and their F-actin coverage and aspect ratio. (d-f) C2C12 cells adhered to MSNFs and showed oriented aligned morphology in each layer within the aligned MSNF/Gel scaffold. Adapted with permission from , copyright 2022, Elsevier. C) Schematics of preparation of an anisotropic hydrogel based on a mussel-inspired conductive ferrofluid. D) Schematic representation of C2C12 cells seeded on the hydrogels and cells being electrically stimulated under different voltages, and CLSM micrographs of C2C12 cells on the anisotropic and isotropic hydrogels after 1 and 3 days of culturing under different electrical stimulation voltages. Adapted with permission from , copyright 2019, American Chemical Society.
Figure 19
Figure 19
A) Schematic illustration of the fabrication procedure of GelMA-GNR microgrooved tissues, and z-Stack images showing the 3D structure of the microgrooved patterns. B) Phase-contrast images of cardiac cells seeded on GelMA and GelMA-GNR microgrooved hydrogel, and fluorescent viability images of GelMA and GelMA-GNR microgrooved cardiac tissues on day 1 and day 7 of culture. C) Immunostained images of SATN (green) and Cx43 gap junctions (red) within GelMA and GelMA-GNR microgrooved cardiac tissues on day 7 of culture, and quantified area coverage of cardiac specific markers on day 7. D) Synchronous spontaneous beating behavior (beats per minute; BPM) of cardiac tissues on GelMA and GelMA-GNR constructs, voltage excitation thresholds, and extracted beating signals of GelMA-GNR cardiac tissue at different frequencies. Adapted with permission from , copyright 2017, Royal Society of Chemistry.
Figure 20
Figure 20
A) Schematic illustration of constructing biomimetic nerve fibers and their neural performances. (a) PC12 cells guided by oriented ECM; (b) RT4-D6P2T cells guided by oriented ECM; (c) Enhanced intercommunication between the two types of cells. B) (a, b) Morphology of PC12 and RT4-D6P2T cells cultured for 7 d. (c, d) Histograms of PC12 and RT4-D6P2T cells length. (e) Axons diameters of PC12 cells counted from immunofluorescence images marked by Tubulin-β3. (f, g) Elisa assays of Tubulin-β3 and SYN1 expressed by PC12 cells and neurotrophic factors including BDNF, CNTF and NGF expressed by RT4-D6P2T cells. (h) Schematic illustration of possible extracellular topographic signaling transduction mechanism. Adapted with permission from , copyright 2020, IOP Publishing.
Figure 21
Figure 21
A) a) Schematic illustration for astrocyte-laden microfibers. b) SEM image of astrocyte-laden microfibers after drying and dehydrating. c) CLSM images of astrocytes encapsulated in the fibers. d) Viabilities of astrocytes encapsulated in the fibers on 2 and 9 days after fabrication. e) CLSM images of astrocytes stained for GFAP and nucleus on 9 days after fabrication. B) a, b) Maximum-intensity-projected CLSM z-stack images of hippocampal neurons on astrocyte-laden microfibers. c) Number of presynapses of a single hippocampal neuron. Adapted with permission from , copyright 2022, Wiley-VCH. C) Schematic illustration for biomimetic construction of HA/Col hydrogel incorporated with viscoelasticity and nano-alignment, as well as the exploration for co-effects of extracellular viscoelasticity and nano-orientation on neuronal cell/tissue behaviors. D) Viability and morphology of PC12 cells. Adapted with permission from , copyright 2021, Elsevier.
Figure 22
Figure 22
A) Schematic representation of preparation of human keratocyte (HK) loaded hydrogels. (a) GelMA slabs, and (b) 3D bioprinted GelMA hydrogels. Adapted with permission from , copyright 2019, Royal Society of Chemistry. B) (a) Schematic illustration of the alignment of collagen fibers within the nozzle during bioink extrusion. (b) Viscosity profile of 2%-Co-dECM bioink incorporating cells. (c) Radial distribution of wall shear stress upon 3D printing for the different-sized nozzles. (d) Second harmonic generation (SHG) images of shear-aligned collagen. (e) Distributions of orientations obtained by analysis using Orientation-J of SHG images collected at different azimuthal angles. C) Cellular behavior of the differentiated keratocytes encapsulated in the bioink on the 28th day of cell culture. Adapted with permission from , copyright 2019, IOP Publishing.
Figure 23
Figure 23
A) MSCs encapsulated in GelMA springs self-organized into smooth muscle-like tissues with cell alignment and contractile property similar to that of vSMCs in the media layer of blood vessels in vivo. B) Formation of MSC springs. (a) Self-organization of MSCs in a GelMA spring. (b) An MSC spring with a large length-to-diameter ratio of about 13.33. (c) Live/dead staining images showing cell viability in the resulting MSC springs encapsulated in GelMA springs with three different diameters. Adapted with permission from , copyright 2020, Royal Society of Chemistry. C) Schematic of fabrication device with resulting macroscopic tubular gel. (a) Empty shear chamber assembled and (b) PA solution loaded in shear chamber. (c) Fabrication procedure showing the inner rod's combined rotation and retraction movement allowing the Ca2+ solution to flow into the lumen of the tube. (d) Macroscopic photo of final PA tube retaining its tubular shape. The aligned sample (e) shows noticeably more aligned nanofibers than the non-aligned sample(f). D) Cellular alignment viewed from inner surface with the tube's long axis in the vertical direction; (a-d) Non-aligned samples, (e-h) Aligned samples. Adapted with permission from , copyright 2012, Elsevier.
Figure 24
Figure 24
A) A graphical illustration of dermis in skin, and SEM image of aligned collagen fiber bundles observed in the dermis of neonatal rat skin and aligned dermal fibroblasts in the same tissue. The orientation of bundles agrees with the tension line in skin. Adapted with permission from , copyright 2012, Elsevier. B) Fluorescence microscopy images and schematic representation of NHDFs culture on highly oriented hydrogels after 48 h. Adapted with permission from , copyrights 2020, Elsevier. C) Schematic representations of the adult IVD and midsagittal cross-section showing anatomical regions. Adapted with permission from , copyright 2020, Frontiers Media. D) Schematic illustration and fabrication process of WF-IVD. (a) Schematic diagram of a typical intervertebral disk to be simulated by WF-IVD. Images of WF-IVD (b) in different directions and (c) under a 1 kg load. (d) Two-component composite fabrication process. Adapted with permission from , copyright 2021, American Chemical Society.
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