(588d) Elucidating the Role of Decellularized Porcine Skeletal Muscle ECM-Silk Composite Systems on Human THP-1-Derived Macrophage Polarization

Traumatic soft tissue injuries often result in permanent loss of skeletal muscle mass known as volumetric muscle loss (VML). This causes a major clinical challenge for military and civilian medicine. Biomaterials can be used to provide the appropriate structural and mechanical properties as well as the release of bioactive and chemotactic signals to address this issue [1, 2]. While it is possible to implant degradable biomaterials at the injury site and see some functional repair, we lack a clear understanding of how the biomaterial can be used to instruct immune cells towards a regenerative phenotype in the skeletal muscle and the role silk-based biomaterials play in this process.

The long-term goal of this work is to engineer decellularized skeletal muscle extracellular matrix (ECM)-silk based natural biomaterials that, upon implantation, modulate in vivo tissue behavior and collective cell function for soft tissue repair and rehabilitation. Specifically, macrophages (myeloid cell lineage, with two very plastic phenotypes, M1 pro-inflammatory and M2 anti-inflammatory) dominate the inflammatory response for muscle regeneration and thus recent work has shown that understanding the role biomaterial formulation has on macrophage phenotype will be critical for improved material design to meet clinical needs [3-6]. Our lab utilizes a silk-decellularized ECM sponge platform, which can be tuned to degrade in vivo over many months. Previous and on-going work has shown that degradation of silk materials is dependent on the enzyme(s) present and it is known that macrophages and fibroblasts at the injury site are largely responsible for enzymatic activities.

Thus, to obtain a better understanding of macrophage response and enzyme secretion in response to the biomaterial formulation, we investigated the polarization of macrophages in response to two separate silk-ECM composite formats. First, 2D culture of human leukemia monocytes (THP-1s) on silk-decellularized skeletal muscle ECM films were evaluated, with decellularized skeletal muscle ECM coated tissue culture plastic, silk only films, and collagen I coated tissue culture plastic as controls. Using previously described methods [7], THP-1 monocytes are driven towards a macrophage phenotype via treatment with phorbol 12-myristate-13-acetate (PMA) to cause the cells to adhere to the culture surface. Then, adhered cells are either treated with LPS lipopolysaccharide (LPS) and interferon gamma (INF-g) or interleukin-4 (IL-4) and interleukin-13 (IL-13) to polarize them towards either M1 or M2 phenotypes, respectively. Through image analysis and western blots, we evaluated the polarization potential of these cells through pan-macrophage markers (CD68, CD11b), markers for M1 (CD80, iNOS – inducible nitric oxide synthase), and for M2 (CD206 - mannose receptor, CD163) at each point in the polarization process on 2D silk-ECM films and necessary positive and negative control substrates to elucidate the influence of silk and ECM independently and combined on the polarization of the THP-1-derived macrophages.

To determine if the observed trends hold in 3D, adherent THP-1s were cultured in 3D silk-ECM sponges, using silk only sponges and 2D tissue culture plastic culture as the controls. Efficiency of polarization in 3D was evaluated by comparing expression of M1 and M2 markers via western blot to the controls. Ongoing work aims to understand how the biomaterial formulation can be leveraged to instruct and direct macrophage phenotype over time. Additionally, gene expression of key enzymes responsible for biomaterial degradation will be tracked by gene expression analysis and correlated with changes in biomaterial elastic modulus over time. These results will inform future work aimed at designing regenerative constructs for applications in skeletal muscle tissue rehabilitation following traumatic injuries, sports-related injuries, and genetic diseases.

[1] K.H. Nakayama, M. Shayan, N.F. Huang, Engineering Biomimetic Materials for Skeletal Muscle Repair and Regeneration, Adv Healthc Mater 8(5) (2019) e1801168.

[2] S.M. Greising, B.T. Corona, C. McGann, J.K. Frankum, G.L. Warren, Therapeutic Approaches for Volumetric Muscle Loss Injury: A Systematic Review and Meta-Analysis, Tissue Eng Part B Rev 25(6) (2019) 510-525.

[3] J.G. Tidball, Regulation of muscle growth and regeneration by the immune system, Nat Rev Immunol 17(3) (2017) 165-178.

[4] M. Juhas, N. Abutaleb, J.T. Wang, J. Ye, Z. Shaikh, C. Sriworarat, Y. Qian, N. Bursac, Incorporation of macrophages into engineered skeletal muscle enables enhanced muscle regeneration, Nat Biomed Eng 2(12) (2018) 942-954.

[5] A. Urciuolo, L. Urbani, S. Perin, P. Maghsoudlou, F. Scottoni, A. Gjinovci, H. Collins-Hooper, S. Loukogeorgakis, A. Tyraskis, S. Torelli, E. Germinario, M.E.A. Fallas, C. Julia-Vilella, S. Eaton, B. Blaauw, K. Patel, P. De Coppi, Decellularised skeletal muscles allow functional muscle regeneration by promoting host cell migration, Sci Rep 8(1) (2018) 8398.

[6] M. Juhas, N. Abutaleb, J.T. Wang, J. Ye, Z. Shaikh, C. Sriworarat, Y. Qian, N. Bursac, Incorporation of macrophages into engineered skeletal muscle enables enhanced muscle regeneration, Nature Biomedical Engineering 2(12) (2018) 942-954.

[7] A.R.D. Reeves, K.L. Spiller, D.O. Freytes, G. Vunjak-Novakovic, D.L. Kaplan, Controlled release of cytokines using silk-biomaterials for macrophage polarization, Biomaterials 73 (2015) 272-283.