Initial In Vitro Development of a Potassium-Based Intra-Articular Injection for Osteoarthritis

Abstract

The long-term goal of this work is to develop a potassium (K⁺)-based intra-articular (IA) injection for osteoarthritis treatment. Within this context, the objectives of this study were to (1) demonstrate that hyperosmolar K⁺ solutions can suppress proinflammatory macrophage activation and (2) evaluate the therapeutic potential of a hyperosmolar K⁺ solution relative to a clinically utilized drug-based (methylprednisolone acetate [MPA]-a corticosteroid) or cell-based (human mesenchymal stem cell [hMSC]) IA injectable. A 3D in vitro model with poly(ethylene glycol) diacrylate hydrogels encapsulated with proinflammatory interferon-gamma (IFN)-stimulated macrophages (M(IFN)s) was utilized. Long-term changes in cell phenotype in response to short-term stimulation (i.e., mimicking an IA injection) were assessed following treatment with 80 mM K⁺ gluconate, hMSCs, or MPA. Addition of 80 mM K⁺ gluconate to culture media significantly reduced iNOS and TNF protein levels in M(IFN)s. Furthermore, short-term stimulation with K⁺ gluconate elicited a significant increase in the anti/proinflammatory cytokine profile in M(IFN)s, a response that is not noticed with either clinically utilized MPA or an hMSC injectable. Hyperosmolar K⁺ solutions are capable of attenuating proinflammatory macrophage activation. Moreover, when evaluated in an in vitro setting mimicking an IA injection, K⁺ performed significantly better than hMSCs or the corticosteroid MPA. Cumulatively, these results support further development and application of a K⁺-based IA injection toward osteoarthritis research.

Keywords: intra-articular injection; macrophage activation; osteoarthritis; potassium.

FIG. 1.

(A) Experimental design mimicking an IA injection in vitro. M(IFN)s were cultured for 5 days in the continued presence of IFN, with 80 mM K⁺ applied only during the first day and then removed. Other IA injection formulations—human MSCs and MPA—were also included to gain a sense of efficacy and benchmark against current OA treatments. An empty PEGDA hydrogel (∼50 kPa stiffness) was provided at the bottom of the culture well to create a surface that more closely matches the stiffness of articular cartilage (aggregate modulus ∼500 kPa) than traditional plastic 2D surfaces (∼1 × 10⁶ kPa). MSC behavior is known to respond to substrate stiffness. Assessments for treatment efficacy included cell lysate levels of several proinflammatory markers, anti-inflammatory markers, and the ratio between anti/proinflammatory profiles. A bar above the protein denotes normalization. (B) Relative protein production of pooled proinflammatory and pooled anti-inflammatory molecules and the ratio between anti/proinflammatory profiles in Raw 264.7 MΦs after 5 days in culture. Treatments were applied only during the first day of culture. *Denotes a significant difference relative to IFN controls. ^#Denotes a significant difference relative to 24-h 80 mM K⁺ gluconate. ⁺Denotes a significant difference relative to 24-h MSCs. PEGDA, poly(ethylene glycol) diacrylate; MSCs, mesenchymal stem cells; MPA, methylprednisolone acetate.