A Collagen-Mimetic Organic-Inorganic Hydrogel for Cartilage Engineering

Abstract

Promising strategies for cartilage regeneration rely on the encapsulation of mesenchymal stromal cells (MSCs) in a hydrogel followed by an injection into the injured joint. Preclinical and clinical data using MSCs embedded in a collagen gel have demonstrated improvements in patients with focal lesions and osteoarthritis. However, an improvement is often observed in the short or medium term due to the loss of the chondrocyte capacity to produce the correct extracellular matrix and to respond to mechanical stimulation. Developing novel biomimetic materials with better chondroconductive and mechanical properties is still a challenge for cartilage engineering. Herein, we have designed a biomimetic chemical hydrogel based on silylated collagen-mimetic synthetic peptides having the ability to encapsulate MSCs using a biorthogonal sol-gel cross-linking reaction. By tuning the hydrogel composition using both mono- and bi-functional peptides, we succeeded in improving its mechanical properties, yielding a more elastic scaffold and achieving the survival of embedded MSCs for 21 days as well as the up-regulation of chondrocyte markers. This biomimetic long-standing hybrid hydrogel is of interest as a synthetic and modular scaffold for cartilage tissue engineering.

Keywords: cartilage tissue engineering; collagen-mimetic peptide; hybrid material; hydrogel; mesenchymal stromal cells; sol-gel.

Figure 1

Synthesis of a PM block (in blue) and 6M1K/6M2K collagen-like peptides.

Figure 2

Silylation of 6M1K/6M2K yielding the 6M-1Si/6M-2Si hybrid collagen-like peptides.

Figure 3

(a) CD spectrum of collagen-like peptides and their hybrid relatives with the calculated Rpn value and (b) schematic representation of PPII and triple helix formation (adapted from [47]).

Figure 4

Cryo-SEM images of hydrogels obtained at 37 °C in DPBS at pH 7.4 for 24 h.

Figure 5

Rheological evaluation of the collagen type I hydrogel and hybrid hydrogels obtained with 0.1 mg/mL NaF and 10 mg/mL glycine at 37 °C in DPBS pH 7.4 and aged for six days. (a) Maximal stress before rupture; (b) maximal strain before rupture; (c) Young’s modulus; (d) mesh size calculated by rheology.

Figure 6

Water re-uptake of freeze-dried hydrogels made with 0.1 mg/mL NaF and 10 mg/mL glycine after 8 and 24 h at 37 °C in DPBS pH 7.4 aged for six days.

Figure 7

Cell viability of hMSCs measured by LIVE/DEAD staining in different hydrogels after one day of encapsulation. Images are the maximum intensity z-projection. Scale bar is 100 μm.

Figure 8

Cryo-SEM images of hydrogels made with 0.1 mg/mL NaF and 10 mg/mL glycine at 37 °C in DPBS pH 7.4 with 10⁶ hMSCs/mL one day after encapsulation.

Figure 9

(a) Cell viability of hMSCs measured by the LIVE/DEAD assay in 6M-2Si/6M-1Si hydrogels after 1 and 21 days of encapsulation. Images are the maximum intensity z-projection. Scale bar is 100 μm. (b) Number of hMSCs measured by DNA quantification in a 6M-2Si/6M-1Si hydrogel at day 0, 1, 7 and 21 days after encapsulation. Data are expressed as the mean ± SEM of four independent experiments and normalized to 100% at day 0. (c) Chondrocyte gene expression in hMSCs encapsulated in 6M-2Si/6M-1Si hydrogels after 21 days of differentiation. Data are expressed as the mean ± SEM of three independent experiments and expressed in a fold change compared with undifferentiated hMSCs at day 0 (dotted line). Statistical analyses were performed using GraphPad prism 9 software with the non-parametric Mann-Whitney test to compare the two conditions. Values are statistically different when p < 0.05 (* p < 0.05).