Design of a Microfiber Scaffold for DAMP Scavenging in Osteoarthritis

Introduction: Osteoarthritis (OA), affecting 32.5 million people as of 2020 (CDC), is increasingly recognized as an inflammatory disease linked to chronic, low-grade inflammation. Damage-associated molecular patterns (DAMPs) in OA, such as cell-free DNA (cfDNA) and degraded cartilage extracellular matrix (ECM) fragments, trigger inflammation and exacerbate cartilage breakdown, correlating with greater disease severity. This study tested the hypothesis that scavenging these pro-inflammatory DAMPs with a cationic scaffold can reduce inflammation. We incorporated cationic PAMAM nanoparticles into an electrospun PLGA/PCL mesh and confirmed that the scaffold retained DAMP scavenging ability, supported chondrocyte viability and proliferation, and effectively bound cfDNA, demonstrating potential for mitigating inflammation and supporting cartilage tissue engineering.

Materials and

Methods: To fabricate the 5:1 PLGA(50/50):PCL microfiber meshes, the polymers were dissolved in acetic acid. Polyamidoamine-G3-cholesterol(5) nanoparticles (PAMAM-chol NPs) were added to the polymer solution at 0.5 or 4 mg/g of scaffold, followed by shaking for one hour and then electrospun using a custom syringe pump onto a grounded plate. The fibers were sputter-coated with a 4 nm layer of gold-palladium and imaged using a Zeiss Sigma VP SEM to assess their morphology. Primary bovine chondrocytes isolated from femoral articular cartilage were incubated with PAMAM-chol NPs at concentrations ranging from 0.01 to 10 μg/mL for 24 hours to assess cytotoxicity based on calcein-AM and EthD-1 staining. Additionally, chondrocytes stimulated with IL-1β were cultured on scaffolds (with and without the highest dose of NP) for two weeks to evaluate the scaffold’s DAMP-scavenging ability in an inflammatory environment. DNA binding was evaluated by incubating the meshes with a DNA stock solution and measuring unbound DNA using a PicoGreen dsDNA assay. Biochemical analysis included characterizing chondrocyte response on scaffolds using the PicoGreen dsDNA assay for cell proliferation, the hydroxyproline assay for collagen deposition, and the dimethylmethylene blue (DMMB) assay for glycosaminoglycan (GAG) deposition. Statistical analyses were performed with GraphPad Prism, using one-way or two-way ANOVA with a Tukey-Cramer post-hoc correction, depending on the number of factors involved.

Results, Conclusions, and Discussions: PAMAM-chol nanoparticles (NPs) slightly reduced cell viability at 10 μg/mL. However, autophagosomes appeared at 1 μg/mL and increased with higher NP doses, indicating potential toxicity (Fig. 1A-C). To minimize systemic exposure, NPs were incorporated into an electrospun mesh (Fig. 1D) and their DNA-binding property measured (Fig. 1E). The NPs retained their scavenging ability within the mesh, showing no reduction in DNA-binding compared to free NPs. Additionally, all NP scaffolds bound significantly more DNA than the control scaffold, which lacked intrinsic scavenging ability.

To assess the anti-inflammatory potential of PAMAM-chol NPs, chondrocytes were seeded on NP scaffolds and stimulated with IL-1β for 72 hours. The cells remained viable, proliferated, and produced collagen and glycosaminoglycans (GAG) (Fig. 1F, G). The scaffold also bound cell-free DNA (cfDNA) released by the cells, as cfDNA per cell was significantly reduced after 72 hours compared to cells seeded on control scaffolds. This reduction was comparable to cells seeded on control scaffolds supplemented with exogenous PAMAM-chol (Fig. 1G).

This study provides preliminary data supporting the use of PAMAM-chol NP-containing scaffolds for DAMP scavenging. However, this study is constrained by limited characterization of cell behavior and short time points. The IL-1β stimulation duration was insufficient, as chondrocytes did not maintain an inflammatory phenotype. Future work will extend the IL-1β stimulation duration and assess the scaffolds' ability to bind other DAMPs, such as HMGB1 or cartilage ECM fragments, and reduce TLR signaling. In summary, these findings highlight the potential of PAMAM-chol NP-containing scaffolds in mitigating inflammation and supporting chondrocyte function, justifying further research into their evaluation in vivo.

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