Improved long-term durability of allogeneic heart valves in the orthotopic sheep model.

OBJECTIVES: Frozen cryopreservation (FC) with the vapour phase of liquid nitrogen storage (-135°C) is a standard biobank technique to preserve allogeneic heart valves to enable a preferable allograft valve replacement in clinical settings. However, their long-term function is limited by immune responses, inflammation and structural degeneration. Ice-free cryopreserved (IFC) valves with warmer storage possibilities at -80°C showed better matrix preservation and decreased immunological response in preliminary short-term in vivo studies. Our study aimed to assess the prolonged performance of IFC allografts in an orthotopic pulmonary sheep model. METHODS: FC (n = 6) and IFC (n = 6) allografts were transplanted into juvenile Merino sheep. After 12 months of implantation, functionality testing via 2-dimensional echocardiography and histological analyses was performed. In addition, multiphoton autofluorescence imaging and Raman microspectroscopy analysis were applied to qualitatively and quantitatively assess the matrix integrity of the leaflets. RESULTS: Six animals from the FC group and 5 animals from the IFC group were included in the analysis. Histological explant analysis showed early inflammation in the FC valves, whereas sustainable, fully functional, devitalized acellular IFC grafts were obtained. IFC valves showed excellent haemodynamic data with fewer gradients, no pulmonary regurgitation, no calcification and acellularity. Structural remodelling of the leaflet matrix structure was only detected in FC-treated tissue, whereas IFC valves maintained matrix integrity comparable to that of native controls. The collagen crimp period and amplitude and elastin structure were significantly different in the FC valve cusps compared to IFC and native cusps. Collagen fibres in the FC valves were less aligned and straightened. CONCLUSIONS: IFC heart valves with good haemodynamic function, reduced immunogenicity and preserved matrix structures have the potential to overcome the known limitations of the clinically applied FC valve.

Effects on human heart valve immunogenicity in vitro by high concentration cryoprotectant treatment.

It has been shown previously that cryopreservation, using an ice-free cryopreservation method with the cryoprotectant formulation VS83, beneficially modulated immune reactions in vivo and in vitro when compared with conventionally frozen tissues. In this study, we assessed the impact of a VS83 post-treatment of previously conventionally frozen human tissue on responses of human immune cells in vitro. Tissue punches of treated and non-treated (control) aortic heart valve tissue (leaflets and associated aortic root) were co-cultured for 7 days with peripheral blood mononuclear cells or enriched CD14+ monocytes. Effects on cellular activation markers, cytokine secretion and immune cell proliferation were analysed by flow cytometry. Flow cytometry studies showed that VS83 treatment of aortic root tissue promoted activation and differentiation of CD14+ monocytes, inducing both up-regulation of CD16 and down-regulation of CD14. Significantly enhanced expression levels for the C-C chemokine receptor (CCR)7 and the human leukocyte antigen (HLA)-DR on monocytes co-cultured with VS83-treated aortic root tissue were measured, while the interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1 release was suppressed. However, the levels of interferon (IFN)γ and tumour necrosis factor (TNF)α remained undetectable, indicating that complete activation into pro-inflammatory macrophages did not occur. Similar, but non-significant, changes occurred with VS83-treated leaflets. Additionally, in co-cultures with T cells, proliferation and cytokine secretion responses were minimal. In conclusion, post-treatment of conventionally cryopreserved human heart valve tissue with the VS83 formulation induces changes in the activation and differentiation characteristics of human monocytes, and thereby may influence long-term performance following implantation. Copyright © 2017 John Wiley & Sons, Ltd.

Impact of T-cell-mediated immune response on xenogeneic heart valve transplantation: short-term success and mid-term failure.

OBJECTIVES: Allogeneic frozen cryopreserved heart valves (allografts or homografts) are commonly used in clinical practice. A major obstacle for their application is the limited availability in particular for paediatrics. Allogeneic large animal studies revealed that alternative ice-free cryopreservation (IFC) results in better matrix preservation and reduced immunogenicity. The objective of this study was to evaluate xenogeneic (porcine) compared with allogeneic (ovine) IFC heart valves in a large animal study. METHODS: IFC xenografts and allografts were transplanted in 12 juvenile merino sheep for 1-12 weeks. Immunohistochemistry, ex vivo computed tomography scans and transforming growth factor-ß release profiles were analysed to evaluate postimplantation immunopathology. In addition, near-infrared multiphoton imaging and Raman spectroscopy were employed to evaluate matrix integrity of the leaflets. RESULTS: Acellular leaflets were observed in both groups 1 week after implantation. Allogeneic leaflets remained acellular throughout the entire study. In contrast, xenogeneic valves were infiltrated with abundant T-cells and severely thickened over time. No collagen or elastin changes could be detected in either group using multiphoton imaging. Raman spectroscopy with principal component analysis focusing on matrix-specific peaks confirmed no significant differences for explanted allografts. However, xenografts demonstrated clear matrix changes, enabling detection of distinct inflammatory-driven changes but without variations in the level of transforming growth factor-ß. CONCLUSIONS: Despite short-term success, mid-term failure of xenogeneic IFC grafts due to a T-cell-mediated extracellular matrix-triggered immune response was shown.