Dexamethasone-loaded, injectable pullulan-poly(ethylene glycol) hydrogels for bone tissue regeneration in chronic inflammatory conditions

Abstract

Chronic inflammation, infection, and fixation stability disrupts bone tissue regeneration by implants. The elevated levels of inflammatory markers and reactive oxygen species (ROS) damage tissues, inhibit osteoblastic differentiation, and promote bone resorption. Activation of local and chronic inflammatory responses due to the implantable biomaterial poses a high risk of implant failure and compromised bone repair in several pathological conditions. Not much progress has been made in the development of biomaterials that can counter inflammation and ROS along with inducing osteogenic activities for managing bone defects/injuries. We have developed, for the first time, injectable polymeric hydrogels by crosslinking oxidized pullulan (OP, 1% w/v) and 8-arm PEG hydrazine (PEG-HY, 10% w/v) using pH-sensitive and dynamic hydrazone linkages at 37 °C in buffer. The hydrogels were loaded with dexamethasone (Dex), an anti-inflammatory corticosteroid and osteogenic inducer, by covalently linking it to PEG-HY by hydrazone linkages, and their morphological, injectability, viscoelastic, self-healing, swelling, and drug-release properties were investigated. The hydrogels provided a pH-sensitive sustained release of PEG-Dex conjugate (3.62 wt%, 9.22 × 10−5 mol of Dex/gram) for 28 days, with 74.54 and 55.15% PEG-Dex conjugate being released at pH 6.5 and 7.4. ABTS assay showed that hydrogels inhibited 68% radicals within 1 h, and treatment with hydrogel releasates inhibited the pro-inflammatory markers, IL-6 and IL-1β, and elevated the anti-inflammatory marker, TGF-β, in murine osteoblast precursor cells (MC3T3-E1). The hydrogels were found suitable for cell encapsulation and they exhibited 110% viability on treatment with releasates. Finally, the osteogenic activities of hydrogels were ascertained by alkaline phosphatase (ALP) activities, alizarin red S staining, and osteogenic gene expressions- RUNX2, Col-I, OPN, and IBSP. Overall, PEG-Dex conjugate released from hydrogels improved the cell viability and proliferation, and induced the osteoblastic differentiation. The hydrogels with their promising antioxidant and anti-inflammatory properties along with the osteogenic activities show a strong potential as an injectable, extracellular matrix (ECM)-mimicking implantable drug-depot for bone repair applications in chronic inflammatory conditions.