Hydrogels that mimic developmentally relevant matrix and N-cadherin interactions enhance MSC chondrogenesis

Abstract

Methacrylated hyaluronic acid (HA) hydrogels provide a backbone polymer with which mesenchymal stem cells (MSCs) can interact through several cell surface receptors that are expressed by MSCs, including CD44 and CD168. Previous studies showed that this 3D hydrogel environment supports the chondrogenesis of MSCs, and here we demonstrate through functional blockade that these specific cell-material interactions play a role in this process. Beyond matrix interactions, cadherin molecules, a family of transmembrane glycoproteins, play a critical role in tissue development during embryogenesis, and N-cadherin is a key factor in mediating cell-cell interactions during mesenchymal condensation and chondrogenesis. In this study, we functionalized HA hydrogels with N-cadherin mimetic peptides and evaluated their role in regulating chondrogenesis and cartilage matrix deposition by encapsulated MSCs. Our results show that conjugation of cadherin peptides onto HA hydrogels promotes both early chondrogenesis of MSCs and cartilage-specific matrix production with culture, compared with unmodified controls or those with inclusion of a scrambled peptide domain. This enhanced chondrogenesis was abolished via treatment with N-cadherin-specific antibodies, confirming the contribution of these N-cadherin peptides to chondrogenesis. Subcutaneous implantation of MSC-seeded constructs also showed superior neocartilage formation in implants functionalized with N-cadherin mimetic peptides compared with controls. This study demonstrates the inherent biologic activity of HA-based hydrogels, as well as the promise of biofunctionalizing HA hydrogels to emulate the complexity of the natural cell microenvironment during embryogenesis, particularly in stem cell-based cartilage regeneration.

Fig. 1.

Hydrogel design to harness cell–matrix and cell–cell interactions. hMSCs were photoencapsulated within hydrogels that present epitopes for interaction with CD44 and CD168 receptors, as well as N-cadherin. Macromers were designed from hyaluronic acid (to bind to receptors), modified with methacrylates for photo cross-linking, and modified with peptides that either mediate N-cadherin binding or act as scrambled sequence controls. Fluorescent images of hMSCs immunostained for CD44, CD168, or N-cadherin (green) surface receptors and nuclei (blue). (Scale bars, 100 μm.)

Fig. 2.

hMSC interactions with CD44 and CD168 influence chondrogenesis. (A) Mean fold change in gene expression (normalized to GAPDH and monolayer cells before encapsulation) of selected chondrogenic markers (COL2 and AGG) of MSCs cultured in HA hydrogels (1.5% MeHA, wt/vol) after 1, 3, 7, or 28 d of in vitro culture in differentiation media with no treatment (control) or after treatment with CD44 (CD44ab) or CD168 (CD168ab) antibodies before encapsulation. (B) GAG and total collagen content normalized by sample wet weight, and (C) equilibrium modulus (E_Y). (D and E) Quantification and images of immunohistochemical staining for CS and COL2 of MSC-laden HA hydrogel constructs after 28 d of in vitro culture. *P < 0.05 vs. control group at the same culture time (n = 4); ^▪P < 0.1 vs. control group (n = 5). (Scale bars, 50 µm.)

Fig. 3.

HA-based presentation of N-cadherin mimetic peptides enhances early hMSC chondrogenesis. Mean fold change in gene expression (normalized to GAPDH and monolayer cells before encapsulation) of selected chondrogenic markers (A) COL2 and (B) AGG of MSCs cultured in HA hydrogels (1.5% MeHA wt/vol, 10% methacrylate consumption with Cadherin or Scrambled peptides) after 1, 3, 7, or 28 d of in vitro culture in differentiation media either untreated (−ab) or with treatment with the N-cadherin antibody before encapsulation (+ab). *P < 0.05 vs. +ab group of the same scaffold at the same culture time (n = 4).

Fig. 4.

N-cadherin biomimetic peptides enhance neocartilage formation by hMSCs in vitro. (A) GAG and total collagen content normalized by sample wet weight, (B) equilibrium modulus (E_Y), and (C and D) immunohistochemical staining results for chondroitin sulfate (CS) and type II collagen (COL2) of MSC-laden HA hydrogel constructs after 28 d of in vitro culture in differentiation media. *P < 0.05 vs. Scrambled or control group (n = 4). (Scale bars, 50 µm.)

Fig. 5.

N-cadherin biomimetic peptides enhance neocartilage formation by hMSCs in vivo. (A) GAG and total collagen content normalized by sample wet weight and (B and C) immunohistochemical staining results for CS and COL2 of MSC-laden HA hydrogel constructs 28 d after s.c. implantation in nude mice. *P < 0.05 vs. Scrambled or control group (n = 4). (Scale bars, 50 µm.)