In vivo efficacy of a polymer layered decellularized matrix composite as a cell honing cardiovascular tissue substitute

Abstract

Cardiovascular surgery involves reconstruction of tissues that are under cyclical mechanical loading, and in constant contact with pulsatile blood flow. Durable biomaterials for such tissue reconstruction are scarce, as they need to be mechanically strong, hemocompatible, and resist structural deterioration from calcification. While homografts are ideal, they are scarce; xenografts are immunogenic and rendered inactive from glutaraldehyde fixation, causing them to calficy and structurally deteriorate over time; decellularized xenografts are devoid of cells, mechanically weak; and synthetic polymeric scaffolds are thrombogenic or too dense to enable host cell infiltration. In this work, we report the in vivo feasibility of a new polymer-decellularized matrix composite material (decellularized bovine pericardium-polycaprolactone: chitosan) fabricated by electrospinning, which is designed to be mechanically strong and achieve programmed host cell honing to integrate into the host. In a rodent and sheep model, this new material was found to be hemocompatible, and enabled host cell infiltration into the polymer and the decellularized matrix core underlying the polymer. Presence of M2 macrophages and several vascular cell types, with matrix remodeling in the vicinity of the cells was observed in the explanted tissues. In summary, the proposed composite material is a novel approach to create in-situ host integrating tissue substitutes, with better non-thrombogenicity, reduced infections and endocarditis, and potentially the ability to grow with the patient and remodeling into a native tissue structure.

Keywords: BP, Bovine Pericardium; Biomaterial; Cardiovascular; Ch, Chitosan; ECM, Extracellular matrix; Extracellular matrix; Glut, Glutaraldehyde; Glut-BP, Glut fixed untreated BP; H&E, Hematoxylin and Eosin; Inflammation; LDH, Lactate dehydrogenase; PCL, Polycaprolactone; Remodeling; SEM, Scanning Electron Microscopy; WBC, White blood cell; α-SMA, α smooth muscle actin cells.

Graphical abstract

Fig. 1

Surgical implantation of the materials in the dorsal subcutaneous region of rats, after which the animals were followed to 1, 4 or 12 weeks and then explanted for histopathological analysis. Bio-Hybrid composite showed increased tissue deposition compared to the untreated and decellularized BP at 12 weeks (image on far right).

Fig. 2

Cellular infiltration in Bio-Hybrid, decellularized BP and glutaraldehyde fixed untreated pericardium explants in a rat subcutaneous implantation model at 1, 4 and 12 weeks in the material-tissue border and the mid-regions. H&E staining of the explants shows higher number of infiltrating cells in the decellularized and Bio-Hybrid samples from 1 to 12 weeks. Mid-region at 12 weeks depicted better repopulation of cells in Bio-Hybrid compared to the other two groups.

Fig. 3

Matrix remodeling of Bio-Hybrid, decellularized BP and glutaraldehyde fixed untreated pericardium explants in a rat subcutaneous implantation model at 1, 4 and 12 weeks. Pentachrome staining of the explants evaluated for cellular infiltration and extracellular matrix degradation showed remodeling of matrix in decellularized and Bio-Hybrid groups with increased deposition of glycosaminoglycans. (Orange, black, and green arrows indicate collagen, elastin and glycosaminoglycans respectively).

Fig. 4

Calcification in Bio-Hybrid, decellularized BP and glutaraldehyde fixed untreated pericardium explants in a rat subcutaneous implantation model at 1, 4 and 12 weeks. Von kossa staining of the explants shows hyper staining of Glut fixed explants at 4 and 12 weeks with few calcium nodules.

Fig. 5

Immunohistochemistry analysis of the Bio-Hybrid, decellularized BP and glutaraldehyde fixed untreated pericardium explants in a rat subcutaneous implantation model at 1, 4 and 12 weeks. iNOS (M1 marker) staining is shown in green and CD163 staining (M2 marker) is shown in red. Bio-Hybrid explants show a mixed M1-M2 response throughout with increasing expression from 1 to 4 weeks. Glut fixed samples show sparse staining in the border regions in all the time points, decellularized BP showing increasing M1 and M2 response over 1–4 weeks with resolution of the M1 response at 12 weeks. The scale bar is indicated in each image and the last panel shows high magnification image of the 12 week explants.

Fig. 6

Juvenile sheep implantation study of the Bio-Hybrid composite patch in the left carotid artery, main pulmonary artery and left atrium for 90 days. Panel I: Implantation of the Bio-Hybrid patch in three locations (left carotid artery, main pulmonary artery and left atrium) showing a ∼2 ​cm x 0.5 ​cm patch, Panel II: explants at 90 days showing absence of thrombi/vegetation/calcification of Bio-Hybrid, with the implant resembling the surrounding native tissue in all the three locations under high and low flow conditions, Panel III: Blood flow and assessment of stenosis in the three locations using color Doppler ultrasound imaging that did not show any disturbance in all three locations, Panel IV: Angiography of carotid arteries, pulmonary artery and left atrium that did not show any presence of thrombus.

Fig. 7

Cellular infiltration in sheep explants from carotid artery, main pulmonary artery and left atrium after 90 days. H&E staining of all the explants showing uniform cellular infiltration in all the locations.

Fig. 8

Matrix remodeling of the sheep explants from carotid artery, main pulmonary artery and left atrium after 90 days. Pentachrome staining of the explants showing predominant deposition of glycosaminoglycans in carotid and pulmonary positions and with less extent in the left atrial explant.

Fig. 9

Calcification of the sheep explants from carotid artery, main pulmonary artery and left atrium after 90 days. Von kossa staining of the explants from all the three locations showing no calcification.

Fig. 10

Immunohistochemistry analysis of the Bio-Hybrid sheep explants (carotid, pulmonary, left atrial explants) after 90 days from one animal. Panel A: Alpha smooth muscle actin (Alpha SMA) staining of explants and right carotid artery (positive control) showing positive staining of SMA positive cells in the control, carotid and pulmonary samples while the left atrial samples show minimal staining, Panel B: Inducible nitric oxide synthase (iNOS) staining of explants and spleen tissue (control) showing most positive staining for M1 macrophages in the carotid samples followed by pulmonary and atrial explants, Panel C: CD163 staining of M2 macrophages of explants and spleen (control) showing similar presence of M2 macrophages in the carotid and pulmonary explants and few cells in the atrial explants, Panel D: Vimentin staining of explants and control (right carotid artery) showing vimentin positive cells in all the samples. Positive control samples are from the same animal.

Fig. 11

Quantification of cellular infiltration in the rat and sheep explants. A) Quantification of number of cells per frame in the untreated BP, decellularized BP and the Bio-Hybrid explants at 1,4 and 12 weeks showing no difference at 1 week, increase in the decellularized BP at 4 weeks and increase in the Bio-Hybrid at 12 weeks in comparison to the untreated BP, and B) Quantification of number of cells/frame in the Bio-Hybrid (material) in comparison to the surrounding native tissue showing no difference between the two regions.

Supplemental Fig. 1

Representative hematoxylin and eosin histological staining of three rat explanted materials in the glutaraldehyde fixed BP (Glutaraldehyde fixed intact BP), decellularized BP, and Bio-Hybrid groups at 1 week (top panel), 4 weeks (middle panel), and 12 weeks (bottom panel). Representative sections show the cross-section of the explants with surrounding tissue..

Supplemental Fig. 2

Representative pentachrome histological staining of three rat explanted materials in the glutaraldehyde fixed BP (Glutaraldehyde fixed intact BP), decellularized BP, and Bio-Hybrid groups at 1 week (top panel), 4 weeks (middle panel), and 12 weeks (bottom panel). Representative sections show the cross-section of the explants with surrounding tissue..

Supplemental Fig. 3

Representative von kossa histological staining of three rat explanted materials in the glutaraldehyde fixed BP (Glutaraldehyde fixed intact BP), decellularized BP, and Bio-Hybrid groups at 1 week (top panel), 4 weeks (middle panel), and 12 weeks (bottom panel). Representative sections show the cross-section of the explants with surrounding tissue..

Supplemental Fig. 4

Scanning electron microscopic images of the12 week Bio-Hybrid cross sectional explants. (A): Low magnification image of the cross section of the explant showing the material and the tissue deposited below and above the explant, (B): High magnification image of the implanted Bio-Hybrid material at the interface showing the white glistening polymer layer..

Supplemental Fig. 5

Juvenile sheep implantation study of the Bio-Hybrid composite patch in the left carotid artery, main pulmonary artery and left atrium for 90 days for the sheep #7563. Panel I: Implantation of the Bio-Hybrid patch in three locations (left carotid artery, main pulmonary artery and left atrium) showing a ∼2 ​cm ∗ 0.5 ​cm patch, Panel II: explants at 90 days showing absence of thrombi/vegetation/calcification of Bio-Hybrid with the implant resembling the surrounding native tissue in all the three locations under high and low flow conditions, Panel III: Blood flow and assessment of stenosis in the three locations using color doppler ultrasound imaging that did not show any disturbance in all three locations, Panel IV: Angiography of carotid arteries, pulmonary artery and left atrium that did not show any presence of thrombus..

Supplemental Fig. 6

Juvenile sheep implantation study of the Bio-Hybrid composite patch in the left carotid artery, main pulmonary artery and left atrium for 90 days for the sheep #7564. Panel I: Implantation of the Bio-Hybrid patch in three locations (left carotid artery, main pulmonary artery and left atrium) showing a ∼2 ​cm ∗ 0.5 ​cthe implant resembling the surrounding native tissue in all the three locations under high and low flow conditions, m patch, Panel II: explants at 90 days showing absence of thrombi/vegetation/calcification of Bio-Hybrid with Panel III: Blood flow and assessment of stenosis in the three locations using color doppler ultrasound imaging that did not show any disturbance in all three locations, Panel IV: Angiography of carotid arteries, pulmonary artery and left atrium that did not show any presence of thrombus..

Supplemental Fig. 7

Cellular infiltration in the sheep explants from carotid artery, main pulmonary artery and left atrium after 90 days, sheep # 7563. H&E staining of the carotid, main pulmonary artery and left atrial explants showing uniform cellular infiltration..

Supplemental Fig. 8

Matrix remodeling in the sheep explants from carotid artery, main pulmonary artery and left atrium after 90 days, sheep # 7563. Pentachrome staining of the carotid, main pulmonary artery and left atrial explants showing predominant deposition of glycosaminoglycans in the pulmonary and left atrial positions..

Supplemental Fig. 9

Cellular infiltration in the sheep explants from carotid artery, main pulmonary artery and left atrium after 90 days, sheep # 7564. H&E staining of the carotid, main pulmonary artery and left atrial explants showing uniform cellular infiltration..

Supplemental Fig. 10

Matrix remodeling in the sheep explants from carotid artery, main pulmonary artery and left atrium after 90 days, sheep # 7564. Pentachrome staining of the carotid, main pulmonary artery and left atrial explants showing predominant deposition of glycosaminoglycans in the carotid and pulmonary positions and less deposition in the left atrial position.

Supplemental Fig. 11

Von kossa staining of the sheep explants from all the three locations in the animal # 7562 showing no calcification..

Supplemental Fig. 12

Von kossa staining of the sheep explants from all the three locations in the animal # 7564 showing no calcification in carotid and left atrial position but some calcification in the native surrounding tissue in the pulmonary position..

Supplementary Fig. 13

Immunohistochemistry analysis of the Bio-Hybrid sheep (animal 1; #7562, animal 2; #7563 and animal 3; #7564) explants after 90 days. Panel A: Alpha smooth muscle actin (Alpha SMA) staining of the carotid, pulmonary and left atrial explants of the three animals showing positive staining of SMA positive cells in the carotid and pulmonary samples while the left atrial samples show minimal staining, Panel B: Inducible nitric oxide synthase (iNOS) staining of the carotid, pulmonary and left atrial explants of the three animals showing most positive staining for M1 macrophages in the carotid samples followed by pulmonary and atrial explants, Panel C: CD163 staining of M2 macrophages in the carotid, pulmonary and left atrial explants of the three animals showing similar presence of M2 macrophages in the carotid and pulmonary explants and few cells in the atrial explants, Panel D: Vimentin staining of the carotid, pulmonary and left atrial explants of the three animals showing vimentin positive cells in all the samples and Panel E: Positive control samples from animal 1 showing alpha SMA and vimentin positive cells in the right carotid explants, iNOS and CD163 positive cells in the spleen explants as a reference..