Skip to main content

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



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.

This is a preview of subscription content, access via your institution.

Figure 1
Figure 2
Figure 3
Figure 4



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


Extracellular matrix


Epidermal growth factor


Enzyme-linked immunosorbent assay


External RNA Control Consortium


Human cryopreserved amniotic membrane
















Living micronized amniotic membrane


Macrophage colony stimulating factor


Matrix metalloproteinase-9


Mesenchymal stem cells


Peripheral blood mononuclear cells


Phosphate buffered saline


Platelet derived growth factor


Prostaglandin E2


Standard error of mean


Transforming growth factor-β1

TNF-α :

Tumor necrosis factor-α


Vascular endothelial growth factor


  1. 1.

    Abraham, A., et al. Machine learning for neuroimaging with scikit-learn. Front. Neuroinform. 8:14, 2014.

    Article  Google Scholar 

  2. 2.

    Anderson, J. M., A. Rodriguez, and D. T. Chang. Foreign body reaction to biomaterials. Semin. Immunol. 20:86–100, 2008.

    Article  Google Scholar 

  3. 3.

    Arnold, L., et al. Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis. J. Exp. Med. 204:1057–1069, 2007.

    Article  Google Scholar 

  4. 4.

    Ashcroft, G. S., et al. Tumor necrosis factor-alpha (TNF-α) is a therapeutic target for impaired cutaneous wound healing. Wound Repair Regen. 20:38–49, 2012.

    Article  Google Scholar 

  5. 5.

    Badylak, S. F., J. E. Valentin, A. K. Ravindra, G. P. McCabe, and A. N. Stewart-Akers. Macrophage phenotype as a determinant of biologic scaffold remodeling. Tissue Eng. A 14:1835–1842, 2008.

    Article  Google Scholar 

  6. 6.

    Baluk, P., et al. TNF-α drives remodeling of blood vessels and lymphatics in sustained airway inflammation in mice. J. Clin. Investig. 119:2954–2964, 2009.

    Google Scholar 

  7. 7.

    Benoit, M., B. Desnues, and J.-L. Mege. Macrophage polarization in bacterial infections. J. Immunol. 181:3733, 2008.

    Article  Google Scholar 

  8. 8.

    Berger, M. L., M. Mamdani, D. Atkins, and M. L. Johnson. Good research practices for comparative effectiveness research: defining, reporting and interpreting nonrandomized studies of treatment effects using secondary data sources: the ISPOR Good Research Practices for Retrospective Database Analysis Task Force Report—Part I. Value Health 12:1044–1052, 2009.

    Article  Google Scholar 

  9. 9.

    Brown, B. N., J. E. Valentin, A. M. Stewart-Akers, G. P. McCabe, and S. F. Badylak. Macrophage phenotype and remodeling outcomes in response to biologic scaffolds with and without a cellular component. Biomaterials 30:1482–1491, 2009.

    Article  Google Scholar 

  10. 10.

    Cooke, M., et al. Comparison of cryopreserved amniotic membrane and umbilical cord tissue with dehydrated amniotic membrane/chorion tissue. J. Wound Care 23:465–476, 2014.

    Article  Google Scholar 

  11. 11.

    Davis, J. S., et al. The use of skin grafts in the ambulatory treatment of ulcers: report of fifty cases. JAMA LXIV:558–560, 1915.

    Article  Google Scholar 

  12. 12.

    Duan-Arnold, Y., et al. Retention of endogenous viable cells enhances the anti-inflammatory activity of cryopreserved amnion. Adv. Wound Care (New Rochelle) 4:523–533, 2015.

    Article  Google Scholar 

  13. 13.

    Ferraro, N. M., W. Dampier, M. S. Weingarten, and K. L. Spiller. Deconvolution of heterogeneous wound tissue samples into relative macrophage phenotype composition via models based on gene expression. Integr. Biol. (Camb.) 9:328–338, 2017.

    Article  Google Scholar 

  14. 14.

    Gibbons, G. W. Grafix, a cryopreserved placental membrane, for the treatment of chronic/stalled wounds. Adv. Wound Care (New Rochelle) 4:534–544, 2015.

    Article  Google Scholar 

  15. 15.

    Guo, X., et al. Modulation of cell attachment, proliferation, and angiogenesis by decellularized, dehydrated human amniotic membrane in in vitro models. Wounds 29:28–38, 2017.

    Google Scholar 

  16. 16.

    Hao, Y., D. H. Ma, D. G. Hwang, W. S. Kim, and F. Zhang. Identification of antiangiogenic and antiinflammatory proteins in human amniotic membrane. Cornea 19:348–352, 2000.

    Article  Google Scholar 

  17. 17.

    Hopkinson, A., et al. Optimization of amniotic membrane (AM) denuding for tissue engineering. Tissue Eng. C 14:371–381, 2008.

    Article  Google Scholar 

  18. 18.

    Huang, G., et al. Accelerated expansion of epidermal keratinocyte and improved dermal reconstruction achieved by engineered amniotic membrane. Cell Transplant. 22:1831–1844, 2013.

    Article  Google Scholar 

  19. 19.

    Jansky, L., P. Reymanova, and J. Kopecky. Dynamics of cytokine production in human peripheral blood mononuclear cells stimulated by LPS or infected by Borrelia. Physiol. Res. 52:593–598, 2003.

    Google Scholar 

  20. 20.

    Johnson, E. L., J. T. Marshall, and G. M. Michael. A comparative outcomes analysis evaluating clinical effectiveness in two different human placental membrane products for wound management. Wound Repair Regen. 2017. doi:10.1111/wrr.12503.

    Google Scholar 

  21. 21.

    Laurent, R., A. Nallet, L. Obert, L. Nicod, and F. Gindraux. Storage and qualification of viable intact human amniotic graft and technology transfer to a tissue bank. Cell Tissue Bank. 15:267–275, 2014.

    Article  Google Scholar 

  22. 22.

    Lavery, L. A., et al. The efficacy and safety of Grafix((R)) for the treatment of chronic diabetic foot ulcers: results of a multi-centre, controlled, randomised, blinded, clinical trial. Int. Wound J. 11:554–560, 2014.

    Article  Google Scholar 

  23. 23.

    Lavin, Y., et al. Tissue-resident macrophage enhancer landscapes are shaped by the local microenvironment. Cell 159:1312–1326, 2014.

    Article  Google Scholar 

  24. 24.

    Law, C. W., Y. Chen, W. Shi, and G. K. Smyth. Voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol. 15:R29, 2014.

    Article  Google Scholar 

  25. 25.

    Leibovich, S. J., and R. Ross. The role of the macrophage in wound repair. A study with hydrocortisone and antimacrophage serum. Am. J. Pathol. 78:71–100, 1975.

    Google Scholar 

  26. 26.

    Litwiniuk, M., and T. Grzela. Amniotic membrane: new concepts for an old dressing. Wound Repair Regen. 22:451–456, 2014.

    Article  Google Scholar 

  27. 27.

    Liu, D., H. Xiong, P. Ning, J. Chen, and W. Lan. In: 2010 3rd International Conference on Biomedical Engineering and Informatics, Vol. 4, pp. 1633–1635, 2010.

  28. 28.

    Lurier, E. B., et al. Transcriptome analysis of IL-10-stimulated (M2c) macrophages by next-generation sequencing. Immunobiology 222(7):847–856, 2017.

  29. 29.

    Magatti, M., et al. Human amnion favours tissue repair by inducing the M1-to-M2 switch and enhancing M2 macrophage features. J. Tissue Eng. Regen. Med. 2016. doi:10.1002/term.2193.

    Google Scholar 

  30. 30.

    Markova, A., and E. N. Mostow. US skin disease assessment: ulcer and wound care. Dermatol. Clin. 30:107–111, ix, 2012.

  31. 31.

    Mirza, R., L. A. DiPietro, and T. J. Koh. Selective and specific macrophage ablation is detrimental to wound healing in mice. Am. J. Pathol. 175:2454–2462, 2009.

    Article  Google Scholar 

  32. 32.

    Mirza, R. E., M. M. Fang, W. J. Ennis, and T. J. Koh. Blocking interleukin-1β induces a healing-associated wound macrophage phenotype and improves healing in Type 2 diabetes. Diabetes 62:2579–2587, 2013.

    Article  Google Scholar 

  33. 33.

    Mirza, R. E., M. M. Fang, E. M. Weinheimer-Haus, W. J. Ennis, and T. J. Koh. Sustained inflammasome activity in macrophages impairs wound healing in Type 2 diabetic humans and mice. Diabetes 63:1103–1114, 2014.

    Article  Google Scholar 

  34. 34.

    Mirza, R., and T. J. Koh. Dysregulation of monocyte/macrophage phenotype in wounds of diabetic mice. Cytokine 56:256–264, 2011.

    Article  Google Scholar 

  35. 35.

    Mosser, D. M., and J. P. Edwards. Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 8:958–969, 2008.

    Article  Google Scholar 

  36. 36.

    Nassiri, S., I. Zakeri, M. S. Weingarten, and K. L. Spiller. Relative expression of proinflammatory and antiinflammatory genes reveals differences between healing and nonhealing human chronic diabetic foot ulcers. J. Investig. Dermatol. 135:1700–1703, 2015.

    Article  Google Scholar 

  37. 37.

    Niknejad, H., G. Paeini-Vayghan, F. A. Tehrani, M. Khayat-Khoei, and H. Peirovi. Side dependent effects of the human amnion on angiogenesis. Placenta 34:340–345, 2013.

    Article  Google Scholar 

  38. 38.

    Parolini, O., and M. Caruso. Review: preclinical studies on placenta-derived cells and amniotic membrane: an update. Placenta 32(Suppl 2):S186–S195, 2011.

    Article  Google Scholar 

  39. 39.

    Regulski, M., et al. A retrospective analysis of a human cellular repair matrix for the treatment of chronic wounds. Ostomy Wound Manag. 59:38–43, 2013.

    Google Scholar 

  40. 40.

    Roh, J. D., et al. Tissue-engineered vascular grafts transform into mature blood vessels via an inflammation-mediated process of vascular remodeling. Proc. Natl Acad. Sci. USA 107:4669–4674, 2010.

    Article  Google Scholar 

  41. 41.

    Sainson, R. C. A., et al. TNF primes endothelial cells for angiogenic sprouting by inducing a tip cell phenotype. Blood 111:4997–5007, 2008.

    Article  Google Scholar 

  42. 42.

    Seabold, S., and P. Josef. Statsmodels: econometric and statistical modeling with Python. In: Proceedings of the 9th Python in Science Conference, pp 57–61, 2010.

  43. 43.

    Sen, C. K., et al. Human skin wounds: a major and snowballing threat to public health and the economy. Wound Repair Regen. Off. Publ. Wound Heal. Soc. Eur. Tissue Repair Soc. 17:763–771, 2009.

    Google Scholar 

  44. 44.

    Sindrilaru, A., et al. An unrestrained proinflammatory M1 macrophage population induced by iron impairs wound healing in humans and mice. J. Clin. Investig. 121:985–997, 2011.

    Article  Google Scholar 

  45. 45.

    Singh, S., et al. Unbiased analysis of the impact of micropatterned biomaterials on macrophage behavior provides insights beyond predefined polarization states. ACS Biomater. Sci. Eng. 2017. doi:10.1021/acsbiomaterials.7b00104.

    Google Scholar 

  46. 46.

    Spiller, K. L., and T. J. Koh. Macrophage-based therapeutic strategies in regenerative medicine. Adv. Drug Deliv. Rev. 2017. doi:10.1016/j.addr.2017.05.010.

    Google Scholar 

  47. 47.

    Spiller, K. L., et al. The role of macrophage phenotype in vascularization of tissue engineering scaffolds. Biomaterials 35:4477–4488, 2014.

    Article  Google Scholar 

  48. 48.

    Talmi, Y. P., L. Sigler, E. Inge, Y. Finkelstein, and Y. Zohar. Antibacterial properties of human amniotic membranes. Placenta 12:285–288, 1991.

    Article  Google Scholar 

  49. 49.

    Tseng, S. C., D. Q. Li, and X. Ma. Suppression of transforming growth factor-beta isoforms, TGF-beta receptor type II, and myofibroblast differentiation in cultured human corneal and limbal fibroblasts by amniotic membrane matrix. J. Cell. Physiol. 179:325–335, 1999.

    Article  Google Scholar 

  50. 50.

    van Putten, S. M., D. T. A. Ploeger, E. R. Popa, and R. A. Bank. Macrophage phenotypes in the collagen-induced foreign body reaction in rats. Acta Biomater. 9:6502–6510, 2013.

    Article  Google Scholar 

  51. 51.

    Wetzler, C., H. Kämpfer, B. Stallmeyer, J. Pfeilschifter, and S. Frank. Large and sustained induction of chemokines during impaired wound healing in the genetically diabetic mouse: prolonged persistence of neutrophils and macrophages during the late phase of repair. J. Investig. Dermatol. 115:245–253, 2000.

    Article  Google Scholar 

  52. 52.

    Willenborg, S., et al. CCR2 recruits an inflammatory macrophage subpopulation critical for angiogenesis in tissue repair. Blood 120:613–625, 2012.

    Article  Google Scholar 

  53. 53.

    Wilshaw, S. P., J. N. Kearney, J. Fisher, and E. Ingham. Production of an acellular amniotic membrane matrix for use in tissue engineering. Tissue Eng. 12:2117–2129, 2006.

    Article  Google Scholar 

  54. 54.

    Wilshaw, S. P., J. Kearney, J. Fisher, and E. Ingham. Biocompatibility and potential of acellular human amniotic membrane to support the attachment and proliferation of allogeneic cells. Tissue Eng. A 14:463–472, 2008.

    Article  Google Scholar 

  55. 55.

    Witherel, C. E., P. L. Graney, D. O. Freytes, M. S. Weingarten, and K. L. Spiller. Response of human macrophages to wound matrices in vitro. Wound Repair Regen. 24:514–524, 2016.

    Article  Google Scholar 

  56. 56.

    Wolbank, S., et al. Impact of human amniotic membrane preparation on release of angiogenic factors. J. Tissue Eng. Regen. Med. 3:651–654, 2009.

    Article  Google Scholar 

  57. 57.

    Wynn, T. A., and L. Barron. Macrophages: master regulators of inflammation and fibrosis. Semin. Liver Dis. 30:245–257, 2010.

    Article  Google Scholar 

  58. 58.

    Xue, J., et al. Transcriptome-based network analysis reveals a spectrum model of human macrophage activation. Immunity 40:274–288, 2014.

    Article  Google Scholar 

  59. 59.

    Zheng, Y., et al. Topical administration of cryopreserved living micronized amnion accelerates wound healing in diabetic mice by modulating local microenvironment. Biomaterials 113:56–67, 2017.

    Article  Google Scholar 

Download references


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.

Ethical Approval

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.

Author information



Corresponding author

Correspondence to Kara L. Spiller.

Additional information

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.

Supplementary material 1 (CSV 10 kb)

Supplementary material 2 (PNG 77 kb)

Supplementary material 3 (TIFF 1015 kb)

Supplementary material 4 (TIFF 979 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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).

Download citation


  • Macrophage
  • Cell–biomaterial interactions
  • Gene expression
  • Inflammation
  • Wound healing