Free-aldehyde neutralized and oligohyaluronan loaded bovine pericardium with improved anti-calcification and endothelialization for bioprosthetic heart valves

Abstract

The number of patients with valvular heart disease is increasing yearly, and valve replacement is the most effective treatment, during which bioprosthetic heart valves (BHVs) are the most widely used. Commercial BHVs are mainly prepared with glutaraldehyde (Glut) cross-linked bovine pericardial or porcine aortic valves, but the residual free aldehyde groups in these tissues can cause calcification and cytotoxicity. Moreover, insufficient glycosaminoglycans (GAGs) in tissues can further reduce biocompatibility and durability. However, the anti-calcification performance and biocompatibility might be improved by blocking the free aldehyde groups and increasing the GAGs content in Glut-crosslinked tissues. In our study, adipic dihydrazide (ADH) was used to neutralize the residual free aldehyde groups in tissues and provide sites to blind with oligohyaluronan (OHA) to increase the content of GAGs in tissues. The modified bovine pericardium was evaluated for its content of residual aldehyde groups, the amount of OHA loaded, physical/chemical characteristics, biomechanical properties, biocompatibility, and in vivo anticalcification assay and endothelialization effects in juvenile Sprague-Dawley rats. The results showed that ADH could completely neutralize the free aldehyde groups in the Glut-crosslinked bovine pericardium, the amount of OHA loaded increased and the cytotoxicity was reduced. Moreover, the in vivo results also showed that the level of calcification and inflammatory response in the modified pericardial tissue was significantly reduced in a rat subcutaneous implantation model, and the results from the rat abdominal aorta vascular patch repair model further demonstrated the improved capability of the modified pericardial tissues for endothelialization. Furthermore, more α-SMA⁺ smooth muscle cells and fewer CD68⁺ macrophages infiltrated in the neointima of the modified pericardial patch. In summary, blocking free-aldehydes and loading OHA improved the anti-calcification, anti-inflammation and endothelialization properties of Glut-crosslinked BHVs and in particularly, this modified strategy may be a promising candidate for the next-generation of BHVs.

Keywords: adipic dihydrazides; anti-calcification; bioprosthetic heart valves; endothelialization; oligo-hyaluronan.

FIGURE 1

General procedure and reaction principle for the treatment of Glut-crosslinked bovine pericardium modification.

FIGURE 2

Qualitative and quantitative analysis of free aldehyde groups in pericardial tissues. The images show the staining results of Schiff base reagents, and purple-red represents the residual aldehyde groups (A–D). The results of quantitative analysis showed that the residual aldehyde groups in the GA group were significantly higher than those in the ADH and OHA groups (E). Quantitative analysis of free amino groups in tissues (F). Qualitative analysis of OHA content in tissues (G).

FIGURE 3

Histological staining and SEM images of the three groups. The images show HE staining (A), Masson’s trichrome staining (B), Alcian blue staining (C) and SEM (D).

FIGURE 4

The water contact angle, biomechanical properties and susceptibility to collagenase of the three groups. The water contact angle of the leaflets decreased significantly in the OHA group compared with those of the GA and ADH groups (A). There was significant improvement in the NPTD group compared with the GA group (D). The tensile modulus of the linear region (B), ultimate tensile stress (C) and ultimate tensile strain (D) among the three groups were no significant difference. The representative stress‒strain curves of each group are presented (E). There was no difference in the relative mass loss among the three groups (F).

FIGURE 5

Results of FTIR spectra (A) and thermal analysis of the three groups of pericardium. Thermogravimetric analysis showed no significant differences among the three groups. (B) The thermal denaturation temperature of the ADH and OHA samples was distinctly improved compared to that of the GA samples (C).

FIGURE 6

The cytocompatibility and hemocompatibility of the three groups. Human umbilical vein endothelial cell (HUVEC) proliferation and morphology were assessed by 6-CDCFDA live cell fluorescent staining. HUVECs almost completely covered the surfaces in the OHA group (C) on day 5, while only a few cells were present on the surfaces in the GA and ADH groups (A, B). The cytotoxicity of the leaflets was determined by MTT assay, and the relative growth ratios of the GA group were significantly lower than those of the ADH and OHA groups (G). Platelet adhesion on the surface of the samples was evaluated by SEM imaging (D–F) and LDH assay (H). LDH levels in the OHA group were significantly lower than those in the GA and ADH groups. There was no significant difference in the hemolysis rate among the three groups (I).

FIGURE 7

Histological analysis of the rat infrarenal abdominal aorta patch repair model. Images show the 4x (A–C) and 20x (D–F) results of HE staining in the three groups, and typical cobblestone-like cells could be observed in the OHA group . Immunofluorescence staining for the endothelial cell marker CD31 (G–I) showed complete CD31⁺ cell coverage in the OHA group.

FIGURE 8

Immunofluorescence staining with α-SMA⁺ antibody and CD68 antibody was performed to observe the infiltration of smooth muscle cells and macrophages in the neointima of the GA, ADH and OHA groups, and the corresponding quantitative analysis was performed at 4 weeks. Representative images of immunofluorescence staining with anti-α-SMA (green) and CD68 (green) antibodies in each group at 4 weeks. Nuclei were counterstained with DAPI. Scale bar, 50 μm (A, B). The number of α-SMA⁺ and CD68⁺ cells in the neointima was quantified at 4 weeks (C, D). Red arrows indicate the locations of positive cells.

FIGURE 9

Gross specimens, histological characteristics and quantitative analysis after subcutaneous implantation for 60 days. Significant calcifications could be observed in the pericardium of the GA group (A), whereas no calcifications were observed in the ADH and OHA groups (B, C). The images show HE staining (D–F). Alizarin red staining showed that the GA group (G) had more calcium deposition (dark red) than the ADH and OHA groups (H, I). The calcium quantitative analysis showed that the calcium content of the ADH and OHA groups was significantly lower than that of the GA group (J).

FIGURE 10

Characterization of the inflammatory response of the samples after subcutaneous implantation for 2 weeks. The images show the immunohistochemical staining of CD68 and CD3 in each group (A–H) and the corresponding quantitative analysis at 2 weeks (I, J).