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Biomaterials

Biomaterials - Poster Session B

Poster B4 - Decellularized porcine dermal hydrogel modulates macrophage phenotype and function

Thursday, October 24, 2024
2:45 PM – 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  As primary responders of the immune system, macrophages are crucial to the success of wound healing and tissue regeneration. Throughout these processes, macrophages display a wide range of functional phenotypes. Implanted foreign biomaterials, such as those used in breast reconstructive surgery, often result in prolonged, macrophage-driven, pro-inflammatory responses[1]. Due to this, the efficacy of the current standard of care, acellular dermal matrix (ADM), is being reevaluated because of the increased risk of infection and seroma when used with breast implants[2]. Injectable decellularized extracellular matrix (dECM) hydrogels, however, have demonstrated success through harnessing the natural structural properties and material components of the ECM, such as collagen, cell adhesion molecules, and growth factors, to promote biocompatibility and implant integration into tissue[3]. Understanding the phenotypic and functional changes in macrophages due to implanted materials and dECM is crucial in streamlining and decreasing the inflammatory response in healing. Our prior in vivo experiments using a decellularized porcine dermal (DPD) hydrogel, a type of dECM, in conjunction with ADM, promoted integration of ADM into tissue and adipocyte expansion compared to standard of care. Additionally, we identified two macrophage subpopulations, CD9HiArg1HiCD301bLo and CD163HiCD38HiCD301bHi, which were found to be reduced one-week post-implantation in DPD. By utilizing in vitro experimentation, we aim to reveal how DPD hydrogel directly modulates these phenotypic changes to better understand the mechanisms that regulate inflammatory responses. We hypothesize that DPD hydrogel alters expression of CD9, CD163, CD301b, and CD206 on anti-inflammatory macrophages, and that secretions from these macrophages stimulate adipocyte differentiation.

Materials and

Methods:  Lyophilized decellularized porcine dermal tissue was ground through a 40 mesh sieve using a Mini Cutting Mill. A solution of 1.25 mg pepsin / mL 0.01 M HCl was used to digest the powder at 15 mg/mL on a stir plate at room temperature for 72 hours. The pre-gel was neutralized with 0.1 M NaOH and then buffered with PBS. The gel was sterilized in 1 mL syringes under a UV light for 30 minutes. J774A.1 macrophages were cultured in RPMI supplemented with 10% FBS and 1% penicillin-streptomycin and were seeded in a 96-well plate at a concentration of 15,000 cells per well. Macrophages were either seeded directly on plastic, or on top of 50 µl of DPD hydrogel and allowed to adhere overnight. The next day, macrophages either received fresh media or were polarized for 24 hours with IL-4 (20 ng/ml or 40 ng/ml) and IL-10 (20 ng/ml or 40 ng/ml). Conditioned media was collected for later experimentation, and cells were fixed. Immunofluorescent imaging was then conducted on markers of interest.

Results, Conclusions, and Discussions: Immunofluorescent imaging confirmed the presence of CD9, CD163, CD206, and CD301b on cells grown on plastic (Fig 1A-B), as well as successful staining for nuclei on macrophages on both plastic and DPD hydrogel (data not shown). Visible staining was reduced on DPD hydrogel due to limited discrepancy between fluorescence intensity of each marker and background hydrogel intensity, thus hindering our ability to accurately compare macrophage phenotype between DPD and control. Comparison of unstimulated and stimulated wells within the control group showed nominal polarization with upregulated CD163, while CD9, CD206, and CD301b were not significantly changed (Fig 1C). Additionally, nuclei count was significantly reduced in stimulation wells.
In subsequent experiments, polarization will be conducted for longer (i.e., 4 days) to obtain a greater amount of upregulation of markers of interest, while lower concentration stimulation treatment will be utilized to reduce cell death. Longer and repeated PBS washes will be utilized to reduce residual conjugated antibodies from DPD hydrogel. Additionally, the antibody incubation will be lengthened for increased staining of macrophages that infiltrated into the hydrogel after seeding. With these improved experimental and staining protocols, we will quantify marker regulation to understand phenotypic and functional changes of subpopulations in response to DPD hydrogel. In these experiments, we expect that collagen fragments in DPD hydrogel will play a key role in promoting reparative macrophage phenotypes, and will lead to increased expression of CD206, CD163, and CD301b and decreased expression of pro-inflammatory CD9. Studies have shown that collagen fragment accumulation in adipose tissue results in CD206-driven macrophage endocytosis of type I collagen, stimulating proliferation of these tissue resident anti-inflammatory macrophages[4]. For future work, we plan to inhibit CD206 to confirm the phenotypic impact of collagen uptake via this receptor; we postulate that inhibiting CD206 prevents these changes. Furthermore, fibro-adipogenic progenitor cells will be cultured in conditioned media from both DPD hydrogel and control groups to understand how macrophage secretion and signaling impacts adipogenesis and immune response to implanted biomaterials.

Acknowledgements (Optional):  

References:
[1] J. M. Anderson, A. Rodriguez, and D. T. Chang, “FOREIGN BODY REACTION TO BIOMATERIALS,” Semin Immunol, vol. 20, no. 2, pp. 86–100, Apr. 2008, doi: 10.1016/j.smim.2007.11.004.
[2] Y. S. Chun et al., “Implant-Based Breast Reconstruction Using Acellular Dermal Matrix and the Risk of Postoperative Complications,” Plastic and Reconstructive Surgery, vol. 125, no. 2, p. 429, Feb. 2010, doi: 10.1097/PRS.0b013e3181c82d90.
[3] W. Zhang, A. Du, S. Liu, M. Lv, and S. Chen, “Research progress in decellularized extracellular matrix-derived hydrogels,” Regenerative Therapy, vol. 18, pp. 88–96, Dec. 2021, doi: 10.1016/j.reth.2021.04.002.
[4] M. Vujičić et al., “A macrophage-collagen fragment axis mediates subcutaneous adipose tissue remodeling in mice,” Proceedings of the National Academy of Sciences, vol. 121, no. 6, p. e2313185121, Feb. 2024, doi: 10.1073/pnas.2313185121.