GelMA Hydrogel Reinforced with 3D Printed PEGT/PBT Scaffolds for Supporting Epigenetically-Activated Human Bone Marrow Stromal Cells for Bone Repair

Abstract

Epigenetic approaches using the histone deacetylase 2 and 3 inhibitor-MI192 have been reported to accelerate stem cells to form mineralised tissues. Gelatine methacryloyl (GelMA) hydrogels provide a favourable microenvironment to facilitate cell delivery and support tissue formation. However, their application for bone repair is limited due to their low mechanical strength. This study aimed to investigate a GelMA hydrogel reinforced with a 3D printed scaffold to support MI192-induced human bone marrow stromal cells (hBMSCs) for bone formation. Cell culture: The GelMA (5 wt%) hydrogel supported the proliferation of MI192-pre-treated hBMSCs. MI192-pre-treated hBMSCs within the GelMA in osteogenic culture significantly increased alkaline phosphatase activity (p ≤ 0.001) compared to control. Histology: The MI192-pre-treated group enhanced osteoblast-related extracellular matrix deposition and mineralisation (p ≤ 0.001) compared to control. Mechanical testing: GelMA hydrogels reinforced with 3D printed poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) scaffolds exhibited a 1000-fold increase in the compressive modulus compared to the GelMA alone. MI192-pre-treated hBMSCs within the GelMA-PEGT/PBT constructs significantly enhanced extracellular matrix collagen production and mineralisation compared to control (p ≤ 0.001). These findings demonstrate that the GelMA-PEGT/PBT construct provides enhanced mechanical strength and facilitates the delivery of epigenetically-activated MSCs for bone augmentation strategies.

Keywords: 3D printing; GelMA; HDAC inhibitor; MI192; bone; epigenetics; hydrogel; tissue engineering.

Figure 1

The effects of MI192 on the proliferation and viability of hBMSCs within GelMA hydrogels. (a) DNA content of MI192-pre-treated/untreated hBMSCs within GelMA hydrogels during basal culture. (b) Merged live/dead staining of encapsulated hBMSCs within GelMA during osteogenic culture (live cells—green; dead cells—red). Scale bars = 50 µm. Data are presented as the mean ± SD (n = 3).

Figure 2

MI192 pre-treatment (48 h) enhances ALPSA in hBMSCs encapsulated in GelMA hydrogels compared with the untreated group after 2 weeks of osteogenic culture. Data are presented as the mean ± SD (n = 3). *** p ≤ 0.001.

Figure 3

Histological staining of MI192-pre-treated hBMSCs encapsulated within GelMA hydrogels after 6 weeks of osteogenic culture. (a) Picrosirius red staining for collagen production in the MI192-pre-treated constructs. (b) Quantitative analysis of picrosirius red collagen staining. (c) Alizarin red staining for calcium deposition with MI192-pre-treated hBMSCs. (d) Quantitative analysis of Alizarin red staining. (e) von Kossa staining for mineral nodules. (f) Semi-quantitative analysis of mineral nodules. Scale bars = 100 µm. Data are presented as the mean ± SD. *** p ≤ 0.001.

Figure 4

Effects of MI192 pre-treatment on Col1a and OCN expression in hBMSCs within GelMA hydrogels after 6 weeks of osteogenic culture. Scale bars = 200 and 50 µm for the low and high magnification, respectively.

Figure 5

Mechanical properties of GelMA, PEGT/PBT and the GelMA–PEGT/PBT construct. (a) Representative images of the GelMA–PEGT/PBT construct following photo-curing. (b) Compressive modulus of the GelMA hydrogel, the PEGT/PBT scaffold and the GelMA–PEGT/PBT construct. Data are presented as the mean ± SD. *** p ≤ 0.001.

Figure 6

Histological staining on the cross-sections of MI192 pre-treated/untreated hBMSCs encapsulated within GelMA–PEGT/PBT constructs after 6 weeks of osteogenic culture. (a) Picrosirius red staining for collagen production in the MI192-pre-treated GelMA–PEGT/PBT constructs. (b) Quantitative analysis of picrosirius red collagen staining. (c) Alizarin red staining for calcium deposition with the MI192-pre-treated GelMA–PEGT/PBT constructs. (d) Quantitative analysis of Alizarin red staining. (e) von Kossa staining for mineral nodules. (f) Semi-quantitative analysis of mineral nodules. Scale bars = 200 µm. Data are presented as the mean ± SD. ** p ≤ 0.01 and *** p ≤ 0.001. Note: the white elliptical spaces within the stained sections are the 3D printed PEGT/PBT fibres.