Synergetic integrations of bone marrow stem cells and transforming growth factor-β1 loaded chitosan nanoparticles blended silk fibroin injectable hydrogel to enhance repair and regeneration potential in articular cartilage tissue

Abstract

The cartilage repair and regeneration show inadequate self-healing capability and have some complications, which are inordinate challenges in clinical therapy. Biopolymeric injectable hydrogels, a prominent type of cell-carrier as well tissue engineering scaffolding materials, establish promising therapeutic potential of stem cell-based cartilage-regeneration treatment. In addition, injectable scaffolding biomaterial should have rapid gelation properties with adequate rheological and mechanical properties. In the present investigation, we developed and fabricated the macromolecular silk fibroin blended with polylysine modified chitosan polymer (SF/PCS) using thermal-sensitive glycerophosphate (GP), which contains effective gelation ability, morphology, porosity and also has enhanced mechanical properties to induce physical applicability, cell proliferation and nutrient exchange in the cell-based treatment. The developed and optimised injectable hydrogel group has good biocompatibility with human fibroblast (L929) cells and bone marrow-derived mesenchymal stem cells (BMSCs). Additionally, it was found that SF/PCS hydrogel group could sustainably release TGF-β1 and efficiently regulate cartilage-specific and inflammatory-related gene expressions. Finally, the cartilage-regeneration potential of the hydrogel groups embedded with and without BMSCs were evaluated in SD rat models under histopathological analysis, which showed promising cartilage repair. Overall, we conclude that the TGF-β1-SF/PCS injectable hydrogel demonstrates enhanced in vitro and in vivo tissue regeneration properties, which lead to efficacious therapeutic potential in cartilage regeneration.

Keywords: articular cartilage; chitosan; hydrogel; silk fibroin; stem cells.

FIGURE 1

Schematic illustration of BMSCs and TGF‐β1 encapsulated SF/PCS injectable hydrogel for cartilage repair application

FIGURE 2

The structural interactions and component phase purity of CS, RSF, PCS and SF/PCS were evaluated by FT‐IR (A) and XRD (B) spectroscopic analyses

FIGURE 3

The morphological and interconnected structure of prepared hydrogel groups with different blending ratios were observed and visualised by SEM analysis (A). Porosity (B), swelling ratio (C) and water uptake ratio (D) of different hydrogel groups were presented

FIGURE 4

The evaluations of compressive stress‐strain curve (A), compressive strength (D) and compressive modulus (C) of the blended SF/PCS hydrogel groups were exhibited to show mechanical ability and suitability of the scaffolding material

FIGURE 5

The rheological measurements (storage modulus and loss modulus) of the prepared hydrogel with different blending ratios in different temperatures from 25°C to 45°C

FIGURE 6

In vitro biodegradation analysis (%) of blended hydrogel groups in PBS medium without (A) and with (B) enzymes (protease XIV and Lysozyme) and (C) examination of TGF‐β1 releasing ability from hydrogel groups

FIGURE 7

In vitro qualitative (A) and quantitative (B and C) evaluations of cell compatibility of hydrogel groups on BMSCs and L929 fibroblast cell lines incubated for 24 hours; (A) Fluorescence microscopic observation of BMSCs and L929 proliferations after 24 hours incubation with different hydrogel groups, (Scale bar = 20 μm) (B) quantitative observation of cell survival rate (%) treated with different hydrogel groups under MTT assay and (C) cell survival rate (%) treated with increasing concentrations of TGF‐β1@SF/PCS hydrogel after 24 hours incubation time

FIGURE 8

In vitro quantitative investigations of gene expression levels of different blended concentrations of hydrogel groups under RT‐qPCR method; The chondrogenesis gene expression level of SOX 9, Aggrecan and COL II (cartilage‐specific genes), IL‐1β and IL‐6 (inflammatory genes) were examined at 2 and 4 weeks of post‐encapsulation

FIGURE 9

Histological (H&E staining) observation of in vivo cartilage defect treated with BMSCs and TGF‐β1 embedded SF/PCS hydrogel groups at 6, 12 and 18 weeks of postoperative period; Magnification = ×40

FIGURE 10

Histological (MTS) observation of in vivo cartilage defect treated with BMSCs and TGF‐β1 embedded SF/PCS hydrogel groups at 6, 12 and 18 weeks of postoperative period; Magnification = ×40

FIGURE 11

Quantitative data analysis of in vivo analyses; (A) volume of freshly formed bone tissue and (B) histopathological score for regenerated tissue of different treatment groups at 6, 12 and 18 weeks of postoperative period; Magnification = ×40