Gelatin Methacryloyl Hydrogels for Musculoskeletal Tissue Regeneration

Abstract

Musculoskeletal disorders are a significant burden on the global economy and public health. Hydrogels have significant potential for enhancing the repair of damaged and injured musculoskeletal tissues as cell or drug delivery systems. Hydrogels have unique physicochemical properties which make them promising platforms for controlling cell functions. Gelatin methacryloyl (GelMA) hydrogel in particular has been extensively investigated as a promising biomaterial due to its tuneable and beneficial properties and has been widely used in different biomedical applications. In this review, a detailed overview of GelMA synthesis, hydrogel design and applications in regenerative medicine is provided. After summarising recent progress in hydrogels more broadly, we highlight recent advances of GelMA hydrogels in the emerging fields of musculoskeletal drug delivery, involving therapeutic drugs (e.g., growth factors, antimicrobial molecules, immunomodulatory drugs and cells), delivery approaches (e.g., single-, dual-release system), and material design (e.g., addition of organic or inorganic materials, 3D printing). The review concludes with future perspectives and associated challenges for developing local drug delivery for musculoskeletal applications.

Keywords: GelMA; drug delivery; gelatin; hydrogel; musculoskeletal tissue.

Figure 3

(A) Incorporation of vascular-derived extracellular matrix (vECM)@BMP-2 into GelMA hydrogels for angiogenic induced bone regeneration. The top panel shows a schematic diagram of GelMA-vECM@BMP-2 composite hydrogels. The middle panel represents the degradation of GelMA and GelMA-vECM hydrogels and the release patterns of BMP-2 from those hydrogels. The bottom panel shows an excellent bone regeneration ability of GelMA-vECM@BMP-2 hydrogel. Adapted with permission from Ref. [74]. Copyright 2022, Willey. (B) Spatiotemporal release of bFGF and BMP-2 from GelMA-based hydrogels. The top panel shows a schematic illustration of the fabrication of bFGF-GelMA (F-G)/BMP-2-MCM (B-M) composite hydrogels. The middle panel represents the release kinetics of bFGF and BMP-2. The bottom panel demonstrates that the dual release of bFGF and BMP-2 (F-G/B-M) in a spatiotemporal manner significantly enhanced bone formation compared to the single release of bFGF(F-G/M) or BMP-2 (G/B-M). Adapted with permission from Ref. [82]. Copyright 2021, Springer Nature.

Figure 4

(A) Antibacterial activity of different substrates on agar plates and corresponding colony forming units (CFUs) against S. aureus and E. coli with or without near infra-red radiation laser at 808 nm; laser density, 1.5 W/cm² for 10 min. n = 3; error bars indicate standard deviation, * p < 0.05, ** p < 0.01. Gel: GelMA + HA-DA, Gel/BNN6: Gel containing nitric oxide donor (N,N′-Di-sec-butyl-N,N′-dinitroso-1,4-phenylenediamine), Gel/GO-βCD-BNN6: Gel containing BNN6 loaded GO-βCD Adopted with permission from Ref. [93]. Copyright 2020, American Chemical Society. (B) Assessing the anti-inflammatory and antibacterial properties of the nAg/HNTs/GelMA hybrid hydrogel in vivo. The panel demonstrates the schematic diagram showing the treatment of infected bone defect, bacterial colonies in the tissue of infected bone defect and the concentrations of inflammatory cytokines detected using ELISA. * p < 0.05, ** p < 0.01, NS: no statistical significance. Adapted with permission from Ref. [95]. Copyright 2020, Elsevier. (C) Antibacterial activity of polyetheretherketone (PEEK), SP@GelMA (GelMA coated SP), SP@MX/GelMA (MXene containing GelMA coated SP) and SP@MX-TOB/GelMA tested against S. aureus and E. coli. The top panel represents bacterial growth kinetics curves cultured on different substrates. The middle panel represents SEM images of S. aureus on the PEEK, SP, SP@MX/GelMA, SP@MX-TOB/GelMA for 1 day, and the inhibition zones of the corresponding substrates. The bottom panel shows morphological changes in E. coli cultured on varying substrates and zone of inhibition to the corresponding materials. Red arrows indicate the membrane disruption and distorted morphology of E. coli, green arrows indicate the S. aureus fragment and red circles point to the inhibition zone. Adapted with permission from Ref. [97]. Copyright 2020, American Chemical Society.

Figure 6

(A) Comparison of printing fidelity achieved by 7.5 wt% GelMA and 7.5 wt% nanocomposite bioinks incorporating 1 wt% LPN (Scale bars: 1 mm); low magnification (top panel) and high magnification (middle panel); bottom panel represents matrix mineralization of HBMSCs-laden 3D-printed LPN-GelMA construct cultured under osteogenic differentiation conditions (left) and media without dexamethasone (right) for 21 days. Adapted with permission from Ref. [51]. Copyright 2019, IOP. (B) Comparing printability of GelMA (i) with GelMA incorporating SrCO₃ nanostructures (ii) which also supports high cell viability of encapsulated MSCs as determined by live (green)/dead (red) staining (iii) matrix mineralization assessed via Alizarin Red staining of mineralized (CaP) nodules (iv) in bioprinted cell-laden Sr-GelMA scaffold. Adapted with permission from Ref. [52]. Copyright 2020, Elsevier. (C) GelMA+ (5 wt% GelMA with 5% gelatin crosslinked using LAP) bioprinted to form a porous cylinder construct (diameter, 2 cm; height, 1 cm) with Saos-2 cells in the bioink before (0 day) (i and iii) and after culture (14 days) (ii and iv). (v) The microscopy image (hematoxylin and eosin staining) of the cylinder cross-section indicates the maintenance of a uniform pattern during tissue formation and (vi) Alizarin Red S staining of printed samples (14 days). The higher-magnification images indicate the top and bottom layers along the height of the cylinder. (vii) Model used for fabricating trifurcated tubular bioprinted constructs and the final printed construct. (viii) normalized ALP activity and (ix) Alizarin Red S staining of samples at different positions along the length of the trifurcated tube after 14 days of culture. GelMA+ (5 wt%), Saos-2 (7.5 × 10⁶ cells/mL) and osteogenic medium were used throughout. Adapted with permission from Ref. [113], Copyright 2020, Creative Commons CC-BY-4.0 license. (D) Left: schematic illustration of coaxial bioprinting techniques using human umbilical vascular endothelial cell (HUVEC)-laden angiogenic bioink (core-bioink) and MC3T3-laden osteogenic bioink (shell bioink) printed via the core and shell nozzle, respectively. Right top: confocal fluorescence micrograph of the core-shell structure on day 3 of culture. HUVECs (encapsulated in the core bioink) were in the center of the filament surrounded by MCT3T3 cells in the shell. HUVECs were labeled in blue using ER-cell tracker, MC3T3 cells were stained in green using Calcein-AM and dead cells were stained in red using ethidium homodimer. Right bottom: relative gene expression analysis of CD31 (angiogenesis) and osteocalcin (osteogenesis) in HUVECs and MC3T3 cells encapsulated in the homogeneous (direct co-culture) and core-shell (indirect co-culture) structures printed using monoaxial or coaxial bioprinting techniques, respectively, on days 7, 14 and 21 of culture. Data are presented as mean values ± standard deviations (n = 4). Significant differences are shown with * p < 0.05, ** p < 0.01, and ns indicates the nonsignificant differences. Adapted with permission from Ref. [114]. Copyright 2022, Wiley.

Figure 1

Number of articles focused on GelMA and GelMA for bone regeneration published per year since 2000 according to PubMed.

Figure 2

(A) Schematic illustration of GelMA synthesis. (B) represents different synthesis processes adapted with their corresponding DS (degree of substitution). The conventional method involves adding a large amount of methacrylic anhydride (MAA), but sequential addition requires less amount of MAA and pH adjustments to achieve higher DS. The other method relies on one-pot synthesis where high DS is achieved. (C) represents different experimental conditions tested and the corresponding reaction times. Letters/numbers in bold represent optimum conditions. (D) The graphs on the left represent pH changes during reaction conditions whereas the graphs on left represent DS as a function of molar concentration of CB (carbonate buffer), initial pH and MAA/gelatin ratio. Error bars represent the relative standard deviation of n = 3. Adapted with permission from Ref. [30]. Copyright 2016, Springer Nature.

Figure 5

(A) Macrophage marker expression on GelMA hydrogels with different stiffnesses (iNOS; M1 marker, Arg-1; M2 marker). This study demonstrated that the soft GelMA hydrogel enhanced M2 polarization in vitro and in vivo. Adapted with permission from Ref. [102]. Copyright 2020, American Chemical Society. (B) Assessing macrophage morphology and phenotype in IL-4 incorporated PEGDA and GelMA hydrogels. The macrophages in PEGDA hydrogels showed clump-like cytoplasmic aggregates of F-actin, whereas macrophages in GelMA showed the presence of a prominent cortical shell. The staining images with M1 surface marker (CD86) and M2 surface marker (CD206) and qPCR of M1-related KRF5 and IL-6 and M2-related STAT6 and IL-10 indicate that the GelMA hydrogel with IL-4 enhanced M2 polarization, compared to the PEDGA-IL hydrogel. Adapted with permission from Ref. [103]. Copyright 2017, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. * p < 0.05, ** p < 0.01.