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Properties of viable lyopreserved amnion are equivalent to viable cryopreserved amnion with the convenience of ambient storage.

ABSTRACT: Human amniotic membrane (AM) has a long history of clinical use for wound treatment. AM serves as a wound protective barrier maintaining proper moisture. AM is anti-inflammatory, anti-microbial and antifibrotic, and supports angiogenesis, granulation tissue formation and wound re-epithelialization. These properties of AM are attributed to its native extracellular matrix, growth factors, and endogenous cells including mesenchymal stem cells. Advances in tissue preservation have helped to overcome the short shelf life of fresh AM and led to the development of AM products for clinical use. Viable cryopreserved amnion (VCAM), which retains all native components of fresh AM, has shown positive outcomes in clinical trials for wound management. However, cryopreservation requires ultra-low temperature storage and shipment that limits widespread use of VCAM. We have developed a lyopreservation technique to allow for ambient storage of living tissues. Here, we compared the structural, molecular, and functional properties of a viable lyopreserved human amniotic membrane (VLAM) with properties of VCAM using in vitro and in vivo wound models. We found that the structure, growth factors, and cell viability of VLAM is similar to that of VCAM and fresh AM. Both, VCAM and VLAM inhibited TNF-? secretion and upregulated VEGF expression in vitro under conditions designed to mimic inflammation and hypoxia in a wound microenvironment, and resulted in wound closure in a diabetic mouse chronic wound model. Taken together, these data demonstrate that VLAM structural and functional properties are equivalent to VCAM but without the constraints of ultra-low temperature storage.

SUBMITTER: Dhall S

PROVIDER: S-EPMC6168127 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Properties of viable lyopreserved amnion are equivalent to viable cryopreserved amnion with the convenience of ambient storage.

Dhall Sandeep S Sathyamoorthy Malathi M Kuang Jin-Qiang JQ Hoffman Tyler T Moorman Matthew M Lerch Anne A Jacob Vimal V Sinclair Steven Michael SM Danilkovitch Alla A

PloS one 20181002 10

Human amniotic membrane (AM) has a long history of clinical use for wound treatment. AM serves as a wound protective barrier maintaining proper moisture. AM is anti-inflammatory, anti-microbial and antifibrotic, and supports angiogenesis, granulation tissue formation and wound re-epithelialization. These properties of AM are attributed to its native extracellular matrix, growth factors, and endogenous cells including mesenchymal stem cells. Advances in tissue preservation have helped to overcome ...[more]

PMID: 30278042

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Project description:IntroductionChronic 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 methodsPrimary 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 discussionGene 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 proinflammatory 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.ConclusionsIn 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.

Dataset Information

Properties of viable lyopreserved amnion are equivalent to viable cryopreserved amnion with the convenience of ambient storage.

Publications

Properties of viable lyopreserved amnion are equivalent to viable cryopreserved amnion with the convenience of ambient storage.

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