Poster P6 - Decellularized meniscus resists a pro-inflammatory macrophage response

Introduction: Biomaterials offer a promising solution for the restoration of damaged tissue and contribute $400B to the worldwide medical device market. The success of these materials is contingent upon induction of a pro-reparative immune response in patients. Adverse reactions lead to inadequate healing and unwarranted pain and inflammation, causing a drastic reduction in the quality of life for patients. Literature has shown that biologically derived materials have better immune responses in patients in comparison to synthetic materials and in particular decellularized extracellular matrices support cell growth and viability. To this point, the Gottardi lab has developed a new naturally sourced extracellular matrix biomaterial that is derived from decellularized porcine meniscus (MEND). MEND is based on the selective removal of elastin to create channels for cellular reinvasion. Previous studies have shown that MEND supports successful integration and regeneration of tracheal cartilage without signs of adverse reaction in an in vivo porcine model. However, the immunomodulatory effects of MEND have not been formally studied. Therefore, the goal of this project is to assess MEND’s ability to support cellular reinvasion and resist an immune response using THP1 derived macrophages. We hypothesize that MEND will not elicit a sustained pro-inflammatory immune response in the presence of macrophages.

Materials and

Methods: MEND Fabrication. Native porcine meniscus was sectioned radially (0.5-1 mm thickness), underwent three freeze thaw cycles, and was then subjected to enzymatic digestion via pepsin and elastase to develop channels for recellularization. Macrophage seeding density optimization. THP1 cells, a monocyte cell line derived from human leukemia, were differentiated into M0 macrophages using phorbol-12-myristate-13-acetate (PMA). To encourage cell invasion, MEND was placed at the bottom of the insert in a Transwell plate, and M0 macrophages were seeded on top suspended in 1% FBS media, with 20% FBS media placed on the bottom to create a serum gradient that drives cells to migrate towards a nutrient rich environment. M0 macrophage invasion was then optimized by testing a variety of cell densities in MEND (25k, 50k, 100k, 200k). Analysis of immune response. After 5 days of invasion, samples were collected for RT-qPCR to test key pro-inflammatory and anti-inflammatory genes (CD80, VEGF, CCR7, TNF, CD206, TIMP3). M0 invaded MEND was compared to plated pro-inflammatory (IFN-y, LPS) and anti-inflammatory (IL-4, IL-13) macrophages as controls, and normalized to plated M0 in technical triplicate.

Results, Conclusions, and Discussions:

Results: Immunofluorescence for CD68, a general macrophage marker, confirmed THP1 monocyte differentiation in plated M0 macrophages, as well as successful polarization to pro-inflammatory and anti-inflammatory macrophages. By DAPI staining, we determined that 100k M0 macrophages per MEND was the optimal seeding density with the best homogenous distribution of cell nuclei throughout the cross-section of the scaffold. RT-qPCR showed that MEND did not induce significant upregulation of pro-inflammatory (CD80, Fig. 1 left) or anti-inflammatory (TIMP3, Fig. 1 right) genes.

Discussion: MEND is uniformly pervaded by channels, supporting M0 macrophage invasion. Based on RT-qPCR, MEND does not drive a pro-inflammatory immune response. Similarly, MEND shows promise in remaining a stable scaffold by also not activating anti-inflammatory polarization. This indicates that MEND promotes cell viability and matrix remodeling because of its stability. To assess the immunomodulatory effects of MEND in more physiologically relevant in vitro environments, we are studying polarization by invading MEND with pro-inflammatory and anti-inflammatory macrophages. In addition, flow cytometry will be used to gain a more sophisticated analysis of macrophage phenotypes in the presence of MEND in each environment. Subcutaneous in vivo studies will be conducted to validate these results when the full immune system is at play.

Conclusion: MEND supports macrophage invasion and does not elicit macrophage polarization, indicating that it is a promising biomaterial solution for cartilage and tissue repair.

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