Chronic wounds remain a major clinical challenge. Human cryopreserved viable amniotic membrane (hCVAM) is among the most successful therapies, but the mechanisms of action remain loosely defined. Because proper regulation of macrophage behavior is critical for wound healing with biomaterial therapies, we hypothesized that hCVAM would positively regulate macrophage behavior in vitro, and that soluble factors released from the hCVAM would be important for this effect.
Materials and Methods
Primary human pro-inflammatory (M1) macrophages were seeded directly onto intact hCVAM or cultured in separation via transwell inserts (Soluble Factors) in the presence of pro-inflammatory stimuli (interferon-γ and lipopolysaccharide) to simulate the chronic wound environment. Macrophages were characterized after 1 and 6 days using multiplex gene expression analysis of 37 macrophage phenotype- and angiogenesis-related genes via NanoString™, and protein content from conditioned media collected at days 1, 3 and 6 was analyzed via enzyme linked immunosorbent assays.
Results and Discussion
Gene expression analysis showed that Soluble Factors promoted significant upregulation of pro-inflammatory marker IL1B on day 1 yet downregulation of TNF on day 6 compared to the M1 macrophage control. In contrast, intact hCVAM, which includes both extracellular matrix, viable cells, and soluble factors, promoted downregulation of pro-inflammatory markers TNF, CCL5 and CCR7 on day 1 and endothelial receptor TIE1 on day 6, and upregulation of the anti-inflammatory marker IL10 on day 6 compared to the M1 Control. Other genes related to inflammation and angiogenesis (MMP9, VEGF, SPP1, TGFB1, etc.) were differentially regulated between the Soluble Factors and intact hCVAM groups at both time points, though they were not expressed at significantly different levels compared to the M1 Control. Interestingly, Soluble Factors promoted increased secretion of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α), while direct contact with hCVAM inhibited secretion of TNF, relative to the M1 Control. Both Soluble Factors and intact hCVAM inhibited secretion of MMP9 and VEGF, pro-inflammatory proteins that are critical for angiogenesis and remodeling, compared to the M1 Control, with intact hCVAM having a stronger effect.
In a simulated pro-inflammatory environment, intact hCVAM has distinct anti-inflammatory effects on primary human macrophages, and direct macrophage contact with intact hCVAM is required for these effects. These findings are important for the design of next generation immunomodulatory biomaterials for wound repair and regenerative medicine that may include living cells, soluble factors, or a controlled drug delivery system.
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Human amniotic membrane
Analysis of variance
Chemokine (C–C motif) ligand 5
Complete RPMI culture medium
Complete RPMI culture medium supplemented with M1-stimulating cytokines
Epidermal growth factor
Enzyme-linked immunosorbent assay
External RNA Control Consortium
Human cryopreserved amniotic membrane
Living micronized amniotic membrane
Macrophage colony stimulating factor
Mesenchymal stem cells
Peripheral blood mononuclear cells
Phosphate buffered saline
Platelet derived growth factor
- SEM :
Standard error of mean
Transforming growth factor-β1
- TNF-α :
Tumor necrosis factor-α
Vascular endothelial growth factor
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The authors would like to thank Yi Arnold-Duan and Matthew Moorman (Osiris Therapeutics, Inc.) for their helpful discussions and technical advice in handling hCVAM. This work was sponsored in part by Osiris Therapeutics, Inc., and by NHLBI Grant Number R01 HL130037 to KLS. CEW is grateful for the US Department of Education Graduate Assistance in Areas of National Need (GAANN) Interdisciplinary Collaboration and Research Enterprise (iCARE) Fellowship.
Conflict of interest
KLS discloses a potential conflict of interest: this study was funded in large part by Osiris Therapeutics, Inc. The study was designed by KLS and CEW, with some input from Osiris with respect to the potential impact of different experiments. Employees from Osiris had no part in interpretation of the study’s results. CEW, TY, MC, and WD declare that they have no conflicts of interest.
De-identified hCVAM samples were provided by Osiris Therapeutics as commercially available materials. De-identified human monocytes were purchased from the University of Pennsylvania Human Immunology Core. As such both human materials are exempt from review by the Institutional Review Board. No animal experiments were conducted for this article.
Kara Spiller is an Assistant Professor in Drexel University’s School of Biomedical Engineering, Science, and Health Systems. Dr. Spiller received Bachelor’s and Master’s Degrees in Biomedical Engineering from Drexel in 2007. As an NSF Graduate Research Fellow, she conducted her doctoral research in the design of semi-degradable hydrogels for the repair of articular cartilage in the Biomaterials and Drug Delivery Laboratory at Drexel and in the Shanghai Key Tissue Engineering Laboratory of Shanghai Jiao Tong University. After completing her Ph.D. in 2010, she conducted research in the design of scaffolds for bone tissue engineering on a Fulbright Fellowship in the Biomaterials, Biodegradables, and Biomimetics (the 3Bs) Research Group at the University of Minho in Guimarães, Portugal. She then conducted postdoctoral studies towards the development of immunomodulatory biomaterials for bone regeneration in the Laboratory for Stem Cells and Tissue Engineering at Columbia University, before joining the Faculty of Drexel in 2013. Her research is funded by grants from the National Science Foundation and the NIH, as well as grants from private foundations and industry. Her research interests include the role of inflammation in regenerative medicine, the design of immunomodulatory biomaterials, and international engineering education.
This article is part of the 2017 CMBE Young Innovators special issue.
Associate Editor Alyssa Panitch oversaw the review of this article.
Electronic supplementary material
Below is the link to the electronic supplementary material. Figure S1 Principal component analysis of principal component 1 (PC1) vs. principal component 2 (PC2), which account for 42.5 and 15% of the variance, respectively. Each treatment is represented by a color, M1 Control: grey, Direct Contact: blue, Soluble Factors: gold, while each donor is represented by a different symbol. Filled markers represent day 1 samples while outlined (no-fill) markers represent day 6 samples. Figure S2 NanoString gene expression analysis of all additional genes that were not differentially expressed. Data are represented as Log2(Value/M1 Control) and as the mean of all experimental replicates (n = 4–9) ± standard error of the mean (SEM). A dotted line at a fold change of 1.0 (or 0 on graphs of Log2-transformed data of values normalized to the M1 Control) on each individual gene represents no change vs. the M1 Control. Figure S3 Differentially expressed gene, IL1B, via mixed-effects regression with treatment and time held as fixed effects and donor as a random effect with ***p < 0.0001. Treatment group with a decreasing slope over time indicates a downregulation over time.
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Witherel, C.E., Yu, T., Concannon, M. et al. Immunomodulatory Effects of Human Cryopreserved Viable Amniotic Membrane in a Pro-Inflammatory Environment In Vitro . Cel. Mol. Bioeng. 10, 451–462 (2017). https://doi.org/10.1007/s12195-017-0494-7
- Cell–biomaterial interactions
- Gene expression
- Wound healing