Hydrostatic Pressure Enhances Chondrogenic Differentiation of Mesenchymal Stem Cells in Silk Fibroin-Based 3D Bioprinted Hydrogels

Abstract

The human meniscus experiences mechanical forces and converts axial loads into hoop stresses. Meniscus injuries and meniscectomies can compromise this function, and therefore, meniscus implants are required. To assess their performance in vitro, it is crucial to recreate a physiological environment. Therefore, we investigated the effect of TGFβ-3-supplemented and TGFβ-free cyclic hydrostatic pressure (HP) up to 10 MPa on 3D bioprinted silk fibroin (SF) polymer-based hydrogels. The bioink was seeded with human infrapatellar fat pad-derived MSCs and supplemented with an extracellular matrix and gelatin. We found that HP stimulation did not alter cell-free biomaterial maturation, while it partially stimulated metabolic activity and cell proliferation. Remarkably, TGFβ-3-supplemented HP led to the highest expression levels of chondrogenic markers, followed by TGFβ-3-supplemented unloaded incubation and then TGFβ-free HP. Despite the low cell density, the combined exposure to TGFβ-3 and HP also facilitated localized glycosaminoglycan and collagen deposition, demonstrating promising prospects for future meniscus regeneration.

1

Biomaterial maturation of printed cell-free controls with three technical replicates. (A) Amide I region in static cell-free controls with the β-sheet region ranging from 1617 to 1627 cm^–1 and the random coil (RC) region from 1640 to 1650 cm^–1 (n = 1). (B) Amide I region in HP cell-free controls (n = 1). (C) Compressive Young’s modulus of static and HP cell-free controls at 10% strain tested by unconfined compression (n = 1). (D) Images of static and HP samples in MSC growth medium.

2

Effect of HP stimulation and application of chondrogenic differentiation medium (TGFβ-3-supplemented) on IFP-MSCs in bioprinted SF10-ECM5-G3 hydrogels on days 0, 14, and 28. Each black dot represents the mean for one biological replicate. (A) Metabolic activity. Data were compared by Kruskal–Wallis tests. No statistical difference was detected (n = 3). (B) Living cell number per μL. Data were compared by Kruskal–Wallis tests. No statistical difference was detected (n = 3). (C) Cell viability. Data were normally distributed by the Shapiro–Wilk test and compared using two-way ANOVA with Tukey post test. No statistical difference was detected (n = 3).

3

Confocal microscopy images after live and dead staining with 4 μM calcein and 8 μM EthD-1 of IFP-MSCs in bioprinted SF10-ECM5-G3 hydrogels on days 0, 14, and 28. The scale bars indicate 300 μm. (A) Biological replicate with IFP-MSCs from donor 1. (B) Biological replicate with IFP-MSCs from donor 2.

4

Gene expression analysis of IFP-MSCs in bioprinted SF10-ECM5-G3 hydrogels for COL1A1, COL2A1, ACAN, SOX9, MMP3 and MMP13. COL10A1 was not expressed in any sample. The Ct values were normalized to GAPDH and to day 0 samples in the MSC growth medium by the 2^–ΔΔCt method. Data was normally distributed (Shapiro–Wilk test) and compared using 2-way ANOVA with Tukey post test. Significance was attributed when p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****). Only the significant differences between days 14 and 28 were displayed in the bar charts. Further comparisons can be found in Table (n = 3).

5

Hematoxylin–Eosin staining of 10 μm thin sections of hydrogels with IFP-MSCs from donor 1. IFP-MSC nuclei are colored dark red to violet, while the background is colored pink. Images were captured at 20× magnification, and the scale bars indicate 100 μm (n = 1).

6

Alcian Blue and Nuclear Fast Red stainings of 10 μm thin sections of hydrogels with IFP-MSCs from donor 1. IFP-MSC nuclei are colored in red, while sGAGs are colored in blue. Images were captured at 20× magnification, and the scale bars indicate 100 μm (n = 1).

7

Azan staining of 10 μm thin sections of hydrogels with IFP-MSCs from donor 1. IFP-MSC nuclei are colored red, while collagen is colored in blue. Images were captured at 20× magnification, and the scale bars indicate 100 μm (n = 1).