A fibrin/hyaluronic acid hydrogel for the delivery of mesenchymal stem cells and potential for articular cartilage repair

Abstract

Background: Osteoarthritis (OA) is a degenerative joint disease affecting approximately 27 million Americans, and even more worldwide. OA is characterized by degeneration of subchondral bone and articular cartilage. In this study, a chondrogenic fibrin/hyaluronic acid (HA)-based hydrogel seeded with bone marrow-derived mesenchymal stem cells (BMSCs) was investigated as a method of regenerating these tissues for OA therapy. This chondrogenic hydrogel system can be delivered in a minimally invasive manner through a small gauge needle, forming a three-dimensional (3D) network structure in situ. However, an ongoing problem with fibrin/HA-based biomaterials is poor mechanical strength. This was addressed by modifying HA with methacrylic anhydride (MA) (HA-MA), which reinforces the fibrin gel, thereby improving mechanical properties. In this study, a range of fibrinogen (the fibrin precursor) and HA-MA concentrations were explored to determine optimal conditions for increased mechanical strength, BMSC proliferation, and chondrogenesis potential in vitro.

Results: Increased mechanical strength was achieved by HA-MA reinforcement within fibrin hydrogels, and was directly correlated with increasing HA-MA concentration. Live/dead staining and metabolic assays confirmed that the crosslinked fibrin/HA-MA hydrogels provided a suitable 3D environment for BMSC proliferation. Quantitative polymerase chain reaction (qPCR) of BMSCs incubated in the fibrin/HA-MA hydrogel confirmed decreased expression of collagen type 1 alpha 1 mRNA with an increase in Sox9 mRNA expression especially in the presence of a platelet lysate, suggesting early chondrogenesis.

Conclusion: Fibrin/HA-MA hydrogel may be a suitable delivery method for BMSCs, inducing BMSC differentiation into chondrocytes and potentially aiding in articular cartilage repair for OA therapy.

Keywords: Cartilage; Fibrin; Hyaluronic acid; Hydrogel; Mesenchymal stem cell; Osteoarthritis; Regenerative medicine; Stem cell delivery; Tissue engineering.

Figure 1

Flow cytometry verifies BMSC phenotype via positive and negative surface markers. Unlabeled BMSCs: (A) FSC vs. SSC and (B-C) Absence of fluorescence detected through any channels. BMSCs treated with CD34-FITC indicate no CD34 expression: (D) FSC vs. SSC, (E) all cells were FITC-negative as detected through channels FL1 and FL2 (100.0% in Q1-LL) and (F) through channels FL3 and FL4 (100.0% in Q2-LL). BMSCs treated with CD105-PE, CD73-PerCP, CD90-APC, CD44-FITC indicate expression of all four surface proteins: (G) FSC vs. SSC, (H) 99.8% of cells (Q1-UR) were PE, PerCP, APC and FITC-positive as detected through FL1 and FL2 and (I) 99.6% of cells (Q2-UR) were detected positive through FL3 and FL4.

Figure 2

¹H NMR spectra of HA (A) and HA-MA (B) with proton peaks in the methyl groups as well as the sugar rings labeled. ¹H for relevant peaks in each figure have been underlined.

Figure 3

BMSC metabolic activity in fibrin/HA-MA hydrogels with varying HA-MA concentrations. (A) 4 mg/mL and (B) 6 mg/mL fibrinogen. * – Statistically significant difference between successive days for a given HA-MA concentration (p < 0.05). † – Statistically significant difference between successive HA-MA concentrations for a given day (p < 0.05).

Figure 4

Representative images of live/dead staining of BMSCs in fibrin/HA-MA hydrogels. Live cells were stained with Calcein-AM (Red) and dead cells with EthD-1 (Green). Formulation with 4 mg/mL fibrinogen with 0 mg/mL HA-MA at (A) day 2, (B) day 4, and (C) day 6. Formulation with 4 mg/mL fibrinogen with 1 mg/mL HA-MA at (D) day 2, (E) day 4, and (F) day 6. Formulation with 6 mg/mL fibrinogen with 1 mg/mL HA-MA at (G) day 2, (H) day 4, and (I) day 6. Scale bar: 100 μm.

Figure 5

Compressive modulus of fibrin/HA-MA hydrogels at 20% strain. *Statistically significant difference between successive HA-MA concentrations for a given fibrinogen concentration (p < 0.05). † – Statistically significant difference between different fibrinogen concentrations for a given HA-MA concentration (p < 0.05).

Figure 6

Scanning electron micrographs of (A) 6 mg/mL fibrinogen with no HA-MA at 5000×, (B) 6 mg/mL fibrinogen with no HA-MA at 10,000×, (C) 6 mg/mL fibrinogen with 1 mg/mL HA-MA at 200x, and (D) 6 mg/mL fibrinogen with 1 mg/mL HA-MA at 5000×. Scale bar: 1 μm.

Figure 7

mRNA expression for BMSCs. (A) collagen type 1 alpha 1 gene and (B) SOX9 gene. All data has been normalized to fibrin/HA-MA with 10% FBS condition. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) is the housekeeping gene. * – Statistical significance from the fibrin/HA-MA with 10% FBS condition (p < 0.05) and † – Statistical significance from the Fibrin with PL condition (p < 0.05).