Immunomodulatory Effects of Human Cryopreserved Viable Amniotic Membrane in a Pro-Inflammatory Environment In Vitro

  • Claire E. Witherel
  • Tony Yu
  • Mark Concannon
  • Will Dampier
  • Kara L. Spiller
Article

Abstract

Introduction

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.

Conclusions

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.

Keywords

Macrophage Cell–biomaterial interactions Gene expression Inflammation Wound healing 

Abbreviations

hAM

Human amniotic membrane

ANOVA

Analysis of variance

CCL5

Chemokine (C–C motif) ligand 5

cRPMI

Complete RPMI culture medium

cRPMI-M1

Complete RPMI culture medium supplemented with M1-stimulating cytokines

ECM

Extracellular matrix

EGF

Epidermal growth factor

ELISA

Enzyme-linked immunosorbent assay

ERCC

External RNA Control Consortium

hCVAM

Human cryopreserved amniotic membrane

IFNG

Interferon-γ

IL4

Interleukin-4

IL8

Interleukin-8

IL10

Interleukin-10

IL1A

Interleukin-1α

IL1B

Interleukin-1β

LPS

Lipopolysaccharide

LMAM

Living micronized amniotic membrane

MCSF

Macrophage colony stimulating factor

MMP9

Matrix metalloproteinase-9

MSCs

Mesenchymal stem cells

PBMCs

Peripheral blood mononuclear cells

PBS

Phosphate buffered saline

PDGFB

Platelet derived growth factor

PGE2

Prostaglandin E2

SEM

Standard error of mean

TGFB1

Transforming growth factor-β1

TNF-α

Tumor necrosis factor-α

VEGF

Vascular endothelial growth factor

Supplementary material

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Supplementary material 1 (CSV 10 kb)
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Supplementary material 2 (PNG 77 kb)
12195_2017_494_MOESM3_ESM.tiff (1015 kb)
Supplementary material 3 (TIFF 1015 kb)
12195_2017_494_MOESM4_ESM.tif (980 kb)
Supplementary material 4 (TIFF 979 kb)

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Copyright information

© Biomedical Engineering Society 2017

Authors and Affiliations

  • Claire E. Witherel
    • 1
  • Tony Yu
    • 1
  • Mark Concannon
    • 1
  • Will Dampier
    • 2
  • Kara L. Spiller
    • 1
  1. 1.School of Biomedical Engineering, Science and Health SystemsDrexel UniversityPhiladelphiaUSA
  2. 2.Department of Microbiology and ImmunologyDrexel University College of MedicinePhiladelphiaUSA

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