Valuable effect of Manuka Honey in increasing the printability and chondrogenic potential of a naturally derived bioink

Abstract

Hydrogel-based bioinks are the main formulations used for Articular Cartilage (AC) regeneration due to their similarity to chondral tissue in terms of morphological and mechanical properties. However, the main challenge is to design and formulate bioinks able to allow reproducible additive manufacturing and fulfil the biological needs for the required tissue. In our work, we investigated an innovative Manuka honey (MH)-loaded photocurable gellan gum methacrylated (GGMA) bioink, encapsulating mesenchymal stem cells differentiated in chondrocytes (MSCs-C), to generate 3D bioprinted construct for AC studies. We demonstrated the beneficial effect of MH incorporation on the bioink printability, leading to the obtainment of a more homogenous filament extrusion and therefore a better printing resolution. Also, GGMA-MH formulation showed higher viscoelastic properties, presenting complex modulus G∗ values of ∼1042 ​Pa, compared to ∼730 ​Pa of GGMA. Finally, MH-enriched bioink induced a higher expression of chondrogenic markers col2a1 (14-fold), sox9 (3-fold) and acan (4-fold) and AC ECM main element production (proteoglycans and collagen).

Keywords: Articular cartilage; Extrusion bioprinting; Manuka honey; Mesenchymal stem cells; Methacrylated gellan gum.

Graphical abstract

Fig. 1

(A) Gelation time for GGMA and GGMA-MH hydrogels with photocuring or a combination of ionical- and photo-crosslinking: Tube inverted vial test at 0, 3 and 10 ​min; (B) Water Uptake study of GGMA and GGMA-MH at different time points (insert: zoom on the first 8h of uptake). Tests were performed in triplicates.

Fig. 2

SEM images, representing cross-section microstructure of (A,B) GGMA and (C,D) GGMA-MH hydrogels at magnifications 35x (A,C) and 100x (B,D). Bars ​= ​500 ​μm; (E) Frequency of pores diameter within the ranges: <100 ​μm, 100–1500 ​μm, 150–200 ​μm, >200 ​μm for GGMA (black) and GGMA-MH (grey). Tests were performed in triplicates.

Fig. 3

(A) Unconfined compression test for GGMA and GGMA-MH gels; (B) Rheological analyses for GGMA and GGMA-MH: temperature sweep test in the temperature range 15–45°C and record of G′ and G″ in LVER at each temperature – Red arrow are pointing at the Sol/Gel transition temperatures; (C) Strain sweep test at 37°C showing G′ and G″ with the increase of strain in the range 0.1–20%; (D) G∗ values with the increase of strain (0.1–20%). Tests were performed in triplicates.

Fig. 4

(A) Bioprinting process for MSCs-loaded GGMA and GGMA-MH bioinks via extrusion onto a DMEM/F12-covered printing bed: the obtained constructs were crosslinked via UV (MA groups bonds) and DMEM/F12 (ionic bonds); (B) Printing process with UV-light; (C) Bioprinted GGMA and GGMA-MH constructs; (D,H) Extrusion of GGMA and GGMA-MH formulations; (E,I) Zoom on the filament extruded before being deposited on the printing bed, red lines showing the change in the diameter over the filament length; (F,J) Phase contrast images of the extruded filaments of GGMA and GGMA-MH; (G,K) GFP images of the extruded filaments of GGMA and GGMA-MH.

Fig. 5

(A–D) Cytocompatibility evaluation of bioprinted construct via confocal microscope 3D images: ReadyProbes assay for MSCs-loaded GGMA and GGMA-MH bioprinted construct at day 1 and day 3 of culture (all the cells are in blue (Hoechst) and dead cells are in red (EthBr)); (E) Analysis of cells metabolic activity with MTS assay at day 1, day 3 and day 7. Tests were performed in triplicates. (F–H) Fluorescent labelling of bioprinted constructs staining Nuclei in blue (DAPI) and cytoskeleton in red (PhRhod): 2D images at days 1, 3 and 7 for GGMA and (I–K) GGMA-MH; 3D images at day 7 for (L) GGMA and (M) GGMA-MH. Bars ​= ​100 ​μm.

Fig. 6

Histology staining of bioprinted constructs after 21 days of culture: H&E staining of (A) GGMA and (B) GGMA-MH; Alcian blue staining GAGs for (C) GGMA and (D) GGMA-MH; PicroSirius Red staining for (E) GGMA and (F) GGMA-MH. Arrows pointing at the high deposition of GAGs and Collagen. Bars ​= ​150 ​μm; (G) GAGs quantification at days 1, 7 and 21 of cell culture. There is a statistical difference between the three time points in each condition (∗∗∗∗p ​< ​0.0001); (H) Gene expression analysis for sox9, col2a1 and acan at day 21 for GGMA-MH (fold change with respect to GGMA). Statistics: ∗∗∗∗p ​< ​0.0001, ∗∗p ​< ​0.01. Tests were performed in triplicates.

Fig. 7

SEM micrographs of GGMA and GGMA-MH samples cross-sections at 21 days post-culture. Scale bars: 10 ​μm.