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. 2022 May 8;10(5):1095. doi: 10.3390/biomedicines10051095

Table 6.

Protein-based scaffolds in heart valve tissue engineering.

Scaffold Types Preparation Methods Results Ref.
Collagen-Based Scaffolds
3D-COL
biological scaffolds
Decellularization by SDS extraction; crosslinking (EDC/NSH); enzymatic treatment to remove elastic fibers. Mechanical properties of 3D-COL controlled by crosslinking degree;
3T3 cells adhere and proliferate on COL scaffolds and infiltrated to depth of about 20 mm after 7 days, and 40 mm after 28 days.
[227]
COL/NRASMC matrix Collagen-cell suspension was cast into silicon rubber wells and cultured in an incubator. Uniform tension, during COL compaction, increases the cell content, stimulates their metabolism and leads to stronger constructs;
NRASMCs are metabolically active proved by the elastin inside and around the COL fibers, and the proteoglycans at their interface.
[228]
3D COL disc scaffolds Molding technique by rapid prototyping with 3D inkjet printer VICs proliferate more on 1% w/v COL than 2% or 5%;
VICs remodel the scaffold and synthesize new matrix
(detection of remodeling enzymes, MMPs and ECM gene expression).
[229]
COL-EL; COL-C4S
heterogenous scaffolds
Molding technique using PTFE molds, followed by freeze drying Good cell proliferation on COL, due to natural cells binding via integrin receptors;
C4S increase the cell metabolic activities;
Low cell proliferation on EL, due to its non-integrinsignaling pathway.
[230]
COL-EL
bilayer scaffolds
Solution casting into PTFE molds; freeze drying, repeated twice to obtain bilayer structure. Bilayer scaffolds have anisotropic bending moduli similar to native valves CDCs prefer COL over EL when proliferating, resulting in asymmetrical cell distribution in the two different layers. [210]
3D COL-EL
hydrogels scaffolds
COL-EL composition: 50% COL, 12% EL, 10% PBS, 28% equal parts of DMEM and FBS; pH 7.5; 37°C; 1 h. 3D COL-EL scaffolds support cell attachment, proliferation and differentiation: after 7 days, VICs double their number and exhibited stable levels of integrin β1 and F-actin expression; VECs have a very good proliferation, but the integrin β1 expression remained low. [231]
3D COL-CH composites scaffolds COL:CH (7:1, w/w)
The composites were seeded with 3 types of cells: SMCs, FIs and ECs.
3D COL-CH have good cells adhesion and support ECs differentiation;
SMCs group—large number of SMCs with dense disordered arrangement; SMC+EC group: large number of scattered
ECs with long shuttle shape.
[232]
COL-HA hybrid scaffolds Crosslinking by EDC/NHS route. Structure similar to fibrosa layer of the valve leaflets;
CDCs attachment not affected by the pore size and stiffness.
[233]
Fibrin-Based Scaffolds
Autologous fibrin-based heart valve scaffolds Molding technique;
In vitro: bioreactor conditioning;
In vivo: implantation in sheep pulmonary trunk (3 months).
In vitro: well-organized structure of “conditioned samples”, aligned OCAs in leaflets; cellular detachment, possible cells death in “control samples”;
In vivo: fibrin scaffolds completely resorbed and replaced by ECM proteins; significant tissue development and cell distribution.
[145,234]
(SC-F)
composites biological valves
Coating DPPV with stem cells-fibrin complex Static condition: 1st day—homogenous distribution of SC;
16th day—cell colony formation in SC-F compared to control (no cell clusters);
Dynamic conditions: starting with the 4th day, floating composite clots at the inner surface of the valve and leaflets are observed.
[235]
Fibrin-based tubular heart valves The tube mounted on a frame with three struts which, upon back-pressure, cause the tube to collapse into three coating “leaflets”. In vitro: excellent performance under hydrodynamic conditions, minimal RF (approx. 5%), excellent values for TGV and EOA;
In vivo (sheep, 2 months): substantial recellularization and no significant change in diameter or mechanical properties.
[236]
Tubular construct sutured at the root circumferential line and at three single points of sinotubular junction. Advantage of one-piece construct manufacturing method without glue;
In vivo (sheep, 3 months): no thrombus formation, calcification or stenosis; formation of ECs confluent monolayer on the valve surface.
[140]
Fibrin-based tube-in-stent heart valves Fibrin gel and HUVCs molded as tube-in-stent form and sewn into a self-expandable nitinol stent. Homogeneous cells distribution throughout the valve;
The simulation of the catheter-based delivery (the valves crimping for 20 min) does not influence the valve mechanical properties or functionality.
[120]
F-ELR
biomimetic heart valves
Multi-step injection molding: the valve wall obtained from F gel and the leaflets from F-ELR gel. Good structure cohesion and functionality (opening/closing cycles); Different cell type localization: the vessel-derived α-SMA negative (leaflets) and α-SMA positive cells (valve wall). [32]
F/PLDL-PLGA anisotropic composites
BioTexValve
Molding of PLDL multifilaments and electrospun
PLGA fibers incorporated within fibrin gel.
Anisotropic Young’s moduli comparable with the native aortic leaflets;
The valve withstands aortic flow/pressure conditions in flow-loop system;
Homogeneous distribution of α-SMA, aligned with the longitudinal direction of the wall and leaflets.
[134]
SF/LDI-PEUU nanofibrous scaffolds SF and LDI-PEUU prepared by electrospinning process. Smooth and porous 3D structure of SF/LDI-PEUU scaffolds with randomly oriented fibers;
Good blood compatibility (hemolysis rate <5%);
HUVECs have spindle-shaped morphology and good spread.
[237]

Abbreviations: C4S—chondroitin-4-sulfate; CDCs—cardiosphere-derived cells; CH—chitosan; DMEM—Dulbecco’s modified Eagle medium; DPPV—decellularized porcine pulmonary valve; ECs—endothelial cells; EDC—1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-hydrochloride; EL—elastin; ELR—elastin-like recombinamer; EOA—effective orifice areas; F—fibrin; FBS—fetal bovine serum; FIs—fibroblasts; HA—hyaluronic acid; HUVECs—human umbilical vein endothelial cells; HUVCs—human umbilical vein cells; LDI-PEUU—L-lysine diisocyanate poly(ester-urethane)urea; MMPs—matrix metalloproteinases; NRASMCs—neonatal rat aortic smooth muscle cells; NSH—N-hydroxysuccinimide; OCAs—ovine carotid artery-derived cells; PBS—phosphate buffered saline; PLDL—poly(L/D,L-lactide); PLGA—poly(lactic-co-glycolic acid); PTFE—polytetrafluoroethylene; RF—regurgitant fractions; SCs—stem cells; SDS—sodium dodecyl sulfate; SF—silk fibrinoin; α-SMA—α-smooth muscle actin; SMCs—smooth muscle cells; 3T3—mouse fibroblasts cells; TVG—transvalvular pressure gradients; VECs—valvular endothelial cells; VICs—valvular interstitial cells.