(M-501) Mesenchymal Stem Cell-laden Chitosan-based Injectable Hydrogel with Cellulose Nanocrystals for Bone Tissue Regeneration

Bone fractures and defects pose a significant challenge since bones are vital for support, movement, and organ protection in the human body. Current treatments for bone loss such as dietary supplements and bone grafts have limitations in terms of effectiveness and invasiveness, respectively. To address these limitations of the current standard of care, we set out to develop an injectable hydrogel that consists of chitosan, cellulose nanocrystals (CNCs), and murine mesenchymal stem cells (mMSCs) as a non-invasive, intraosseous injection to promote bone formation.

Chitosan (CS)-based hydrogels are an attractive bone scaffold material for bone tissue engineering. However, it lacks adequate mechanical strength and durability to support the regeneration of load-bearing bone defects. Our central hypotheses are that the incorporation of CNCs will 1) enhance the mechanical integrity of CS-based hydrogels and 2) promote osteogenic differentiation of bone marrow-derived mesenchymal stem cells to support optimal bone healing and formation. In this study, we assessed the structural properties of the CS and CS-CNC hydrogel using rheology. We also investigated the effects of CNCs on osteogenesis and cell differentiation using Alkaline Phosphate (ALP) Activity Assays and Quant-iT™ PicoGreen™ dsDNA Assays.

The main components of the injectable hydrogel are 3% w/v Chitosan (CS) and two gelling agents, 1M β-Glycerophosphate (β-GP) and 25 mg/mL 2-Hydroxyethyl cellulose (HEC). Murine MSCs (mMSCs) were extracted from the femur and tibia of Balb/c mice and cultured. CNCs were synthesized using sulfuric acid hydrolysis for incorporation into the hydrogel.

The hydrogel formulations were prepared using a three-syringe luer-lock system. In brief, CS and β-GP solutions were combined, and then, this mixture was mixed homogeneously with a third syringe containing HEC, serum-free DMEM, and five million mMSCs, with or without CNCs (Figure 1A). To assess the rheological properties of our hydrogels, we used a DHR-3 rheometer with an 8 mm Sandblasted Peltier Plate. Viscosity was measured using a logarithmic sweep and gelation kinetics were measured using a thermal cure procedure at 37°C. To investigate the osteogenic differentiation potential of our cell-laden hydrogels, hydrogels were prepared as previously mentioned. Following preparation, the hydrogel was injected into syringe molds and allowed to gel in a 5% CO₂ incubator at 37°C for 30 minutes. Thereafter, the hydrogel scaffolds were transferred into osteogenic media cultured for 21 days.

At days 7, 14, and 21, cell proliferation was measured using the Quant-iT PicoGreen dsDNA Assay kit, and in vitro osteogenic differentiation was assessed using Biovision’s Alkaline Phosphate Colorimetric Assay kit.

Rheological properties of the hydrogels with and without CNCs were investigated to determine viscosity and gelation kinetics. These properties are significant to decide if the hydrogel is injectable and able to maintain its shape and structure. Our viscosity results demonstrate that both hydrogel formulations show shear-thinning properties as the shear rate increases, owing to the injectability of both CS and CS-CNC hydrogels. Furthermore, all hydrogels demonstrated instantaneous gelation (< 7 s) at physiological conditions, and all hydrogels continued to stiffen following gelation.

In vitro cell proliferation studies demonstrated that the hydrogel formulation with CNCs elicited significantly greater cell proliferation at all timepoints analyzed over three weeks. For both hydrogels, the most DNA was present at day 7. The DNA number was above 50 ng/mg scaffold for the hydrogel containing CNCs and remained high at all time points analyzed; whereas there was a significant decrease in DNA number for the hydrogel without CNCs from day 7 to 21 (Figure 1B). Similarly, the ALP activity in hydrogel formulations with CNCs was significantly greater at all timepoints in comparison to CS hydrogels, respectively (Figure 1C).

This data shows that the CS-CNC hydrogels promote cell proliferation and osteogenic differentiation of mMSCs, which is required for optimal bone healing treatment. Future studies will investigate incorporating growth factors and other bioactive molecules to further enhance the osteogenic properties and potential efficacy outcomes for bone regeneration in vivo. Collectively, our findings hold promise for the development of a novel treatment approach for patients with bone defects, offering improved effectiveness and reduced invasiveness compared to existing options. Broader implications of this project would improve the quality of life for many patients that are suffering from bone-related disorders.