Introduction:: Guided bone regeneration (GBR) is a process used to treat alveolar bone loss. GBR uses resorbable barrier membranes to prevent soft tissue infiltration into areas of new bone growth. To enhance healing, strategies to influence the bioactivity of barrier membranes are being investigated, including the local delivery of active compounds. Electrospun chitosan membranes (ESCMs) have shown promise for enhanced GBR due to drug loading capabilities, biocompatibility, degradation, and pro-healing properties [1]. Macrophage polarization is another strategy that can be implemented to facilitate the healing process. During wound healing, macrophages polarize from a pro-inflammatory phenotype (M1) to a pro-healing phenotype (M2). Raspberry Ketone (RK) is a phenolic compound of red raspberry that possesses anti-inflammatory properties and has shown significant potential in the promotion of macrophage polarization toward the M2, pro-healing, phenotype [2]. This study aimed to evaluate the immunomodulatory and pro-healing effects of RK on RAW 264.7 cells using a microarray for simultaneous analyses of M1 and M2 cytokines when released from ESCMs in vitro.
Materials and Methods:: RAW 264.7 cells (ATCC-TIB-71), a murine derived macrophage-like cell line, were used. Cells were seeded in 24 well plates at 100,00 cell/ml and incubated in DMEM supplemented with 10% FBS and 1% penicillin, streptomycin, and neomycin (Thermofisher, MA) for 24 hours. After 24 hours, M1 polarization was induced by incubating with medium containing 1 µg/mL lipopolysaccharide (LPS). To gain experience with the microarray, a preliminary experiment was conducted. After 24 hours of LPS incubation, medium was replaced with DMEM, DMEM containing 300 ng/ml Prostaglandin E2 (PGE2), or DMEM containing 60 μg/ml RK. On days 1, 2, and 3 after initial LPS stimulation, groups (n=1) were assayed for nitrite concentration (Invitrogen Griess Reagent Kit, CA), and a microarray (RayBiotech Mouse Cytokine Array Q1, GA) evaluated 20 inflammatory cytokines in the culture media. For the primary study, after 24 hours of LPS incubation, medium was replaced with complete DMEM, DMEM containing 300 ng/ml PGE2, or RK loaded membranes in transwells. Transwells were 3 µm pore size inserts and contained 10 mm diameter hexanoic anhydride modified ESCMs loaded with 0, 50, 100, 250, or 500 µg/membrane RK. Positive controls only received 1 µg/mL LPS stimulation, and negative controls did not receive LPS stimulation, RK, or PGE2. Groups (n=3) were assayed on days 1, 3, and 5 for nitrite concentration and cell viability (CellTiter-Glo Luminescent Cell Viability Assay, WI) after treatment. Microarray analysis evaluated 20 inflammatory cytokines in the cell culture media on days 1, 3, and 5 after treatment.
Results, Conclusions, and Discussions:: For preliminary results (n=1) without membrane incorporation, nitrite concentration decreased over time for all groups. There was a decrease in pro-inflammatory cytokines RANTES, MCP-1, IL-12, IL-1β, TNF-α, and IL-6 over time when 60 µg/mL RK or 300 ng/mL PGE2 was added directly to cell culture medium. RK treated groups saw an accelerated decrease of MCP-1 (Fig. 1) and RANTES in comparison to PGE2 treated groups. Additionally, RK treated groups saw a comparable decrease in IL-2 (Fig. 2) and TNF-α in comparison to PGE2 treated groups. Anti-inflammatory cytokines VEGF (Fig. 3) and IL-9 increased over time when RK or PGE2 was added directly to culture medium. The RK treated group had a similar increase in VEGF as compared to PGE2 but not as large an increase in IL-9. Analysis of membrane incorporated groups are in progress. Data suggests that RK decreases pro-inflammatory cytokines and increases anti-inflammatory, pro-healing cytokines. Microarray analysis allowed for the evaluation of multiple factors associated with macrophage polarization. Additionally, Microarray results from preliminary experiments confirmed prior results based on single cytokine assays for IL-1β and TNF-α, indicating that RK has positive effects on polarizing macrophages. The ability to locally deliver RK may have potential for reducing inflammation and stimulating healing for GBR and related tissue engineering strategies.
Acknowledgements (Optional): : This work was supported by a grant from NIDCR R01DE026759 and was in partial fulfillment of the BME undergraduate honors thesis at the University of Memphis.
References (Optional): : [1] Su, H, et al. Biom Mat. 2016;12(1):015003 [2] Kurakula M, et al. Soc for Biom. 2021
