Covalently Grafted Peptides to Decellularized Pericardium: Modulation of Surface Density.

The covalent functionalization of synthetic peptides allows the modification of different biomaterials (metallic, polymeric, and ceramic), which are enriched with biologically active sequences to guide cell behavior. Recently, this strategy has also been applied to decellularized biological matrices. In this study, the covalent anchorage of a synthetic peptide (REDV) to a pericardial matrix decellularized via Schiff base is realized starting from concentrated peptide solutions (10-4 M and 10-3 M). The use of a labeled peptide demonstrated that as the concentration of the working solution increased, the surface density of the anchored peptide increased as well. These data are essential to pinpointing the concentration window in which the peptide promotes the desired cellular activity. The matrices were extensively characterized by Water Contact Angle (WCA) analysis, Differential Scanning Calorimetry (DSC) analysis, geometric feature evaluation, biomechanical tests, and preliminary in vitro bioassays.

Vascular Polyurethane Prostheses Modified with a Bioactive Coating-Physicochemical, Mechanical and Biological Properties.

This study describes a method for the modification of polyurethane small-diameter (5 mm) vascular prostheses obtained with the phase inversion method. The modification process involves two steps: the introduction of a linker (acrylic acid) and a peptide (REDV and YIGSR). FTIR and XPS analysis confirmed the process of chemical modification. The obtained prostheses had a porosity of approx. 60%, Young's Modulus in the range of 9-11 MPa, and a water contact angle around 40°. Endothelial (EC) and smooth muscle (SMC) cell co-culture showed that the surfaces modified with peptides increase the adhesion of ECs. At the same time, SMCs adhesion was low both on unmodified and peptide-modified surfaces. Analysis of blood-materials interaction showed high hemocompatibility of obtained materials. The whole blood clotting time assay showed differences in the amount of free hemoglobin present in blood contacted with different materials. It can be concluded that the peptide coating increased the hemocompatibility of the surface by increasing ECs adhesion and, at the same time, decreasing platelet adhesion. When comparing both types of peptide coatings, more promising results were obtained for the surfaces coated with the YISGR than REDV-coated prostheses.

Multifunctional Gene Carriers Labeled by Perylene Diimide Derivative as Fluorescent Probe for Tracking Gene Delivery.

Multifunctional carriers with both gene transfection property and fluorescent tracking function have attracted significant attention in recent years. Herein, a kind of perylene diimide derivative (PDI-C10C8) is conjugated onto the polyethylenimine-g-poly(lactide-co-glycolide)-g-polyethylenimine (PLGA-PEI) polymer to obtain fluorescent multifunctional polymer and micelles (abbreviated as MP). Then, the REDV-G-TAT-G-NLS (TP-G) peptide sequence is grafted onto this MP to obtain multifunctional micelles labeled by perylene diimide derivative (MP-TP-G). These micelles exhibit enhanced photobleaching stability compared with the reference Cy5-labeled micelles, and the fluorescent images of cellular uptake show bright red emission without any background noise. Confocal laser scanning microscope (CLSM) experiments show that gene complexes can deliver gene into nucleus. MP-TP-G carriers do not enter into the cell nucleus, which proves that the nuclear localization signal sequence may not exert its nucleus accumulation ability via conjugating to the amphiphilic polymers. The high transfection efficiency and the enhanced photobleaching stability, combined with the ability to monitor the detailed process of cellular uptake and gene delivery, make these multifunctional micelles have great potential application for endothelialization of artificial blood vessels and gene delivery process study.

Oligohistidine and targeting peptide functionalized TAT-NLS for enhancing cellular uptake and promoting angiogenesis in vivo.

BACKGROUND: Gene therapy has been developed and used in medical treatment for many years, especially for the enhancement of endothelialization and angiogenesis. But slow endosomal escape rate is still one of the major barriers to successful gene delivery. In order to evaluate whether introducing oligohistidine (Hn) sequence into gene carriers can promote endosomal escape and gene transfection or not, we designed and synthesized Arg-Glu-Asp-Val (REDV) peptide functionalized TAT-NLS-Hn (TAT: typical cell-penetrating peptide, NLS: nuclear localization signals, Hn: oligohistidine sequence, n: 4, 8 and 12) peptides with different Hn sequence lengths. pEGFP-ZNF580 (pZNF580) was condensed by these peptides to form gene complexes, which were used to transfect human umbilical vein endothelial cells (HUVECs). RESULTS: MTT assay showed that the gene complexes exhibited low cytotoxicity for HUVECs. The results of cellular uptake and co-localization ratio demonstrated that the gene complexes prepared from TAT-NLS-Hn with long Hn sequence (n = 12) benefited for high internalization efficiency of pZNF580. In addition, the results of western blot analysis and PCR assay of REDV-TAT-NLS-H12/pZNF580 complexes showed significantly enhanced gene expression at protein and mRNA level. Wound healing assay and transwell migration assay also confirmed the improved proliferation and migration ability of the transfected HUVECs by these complexes. Furthermore, the in vitro and in vivo angiogenesis assay illustrated that these complexes could promote the tube formation ability of HUVECs. CONCLUSION: The above results indicated that the delivery efficiency of pZNF580 and its expression could be enhanced by introducing Hn sequence into gene carriers. The Hn sequence in REDV-TAT-NLS-Hn is beneficial for high gene transfection. These REDV and Hn functionalized TAT-NLS peptides are promising gene carriers in gene therapy.

Multitargeting Gene Delivery Systems for Enhancing the Transfection of Endothelial Cells.

Gene therapy demonstrates promising prospects on cardiovascular diseases. However, nonviral gene delivery system has relatively low transfection efficiency, especially for endothelial cells (ECs). Herein, typical cell-penetrating peptide (TAT), nuclear localization signals (NLSs), and REDV functional peptide have been used to prepare multitargeting complexes. These complexes exhibit higher transfection efficiency owing to the targeting sequences of REDV and NLSs as well as the cell-penetrating function of TAT. The multifunction of the complexes provides high cell uptake, endo/lysosomal escape, and nucleus accumulation of the encapsulated DNA. Thus these multitargeting complexes can provide a potential platform for gene delivery, especially for EC transfection.

Characterization and promotion of endothelialization of Bombyx mori silk fibroin functionalized with REDV peptide.

In the development of small-diameter vascular grafts, it is crucial to achieve early-stage endothelialization to prevent thrombus formation and intimal hyperplasia. Silk fibroin (SF) from Bombyx mori is commonly used for such grafts. However, there is a need to expedite endothelialization post-implantation. In this study, we functionalized SF with Arg-Glu-Asp-Val (REDV) (SF + REDV) using cyanuric chloride to enhance endothelialization. The immobilization of REDV onto SF was confirmed and the amount of immobilized REDV could be calculated by 1H NMR. Furthermore, the conformational changes in Tyr, Ser, and Ala residues in [3-13C]Tyr- and [3-13C]Ser-SF due to REDV immobilization were monitored using 13C solid-state NMR. The REDV immobilized onto the SF film was found to be exposed on the film's surface, as confirmed by biotin-avidin system. Cell culture experiments, including adhesiveness, proliferation, and extensibility, were conducted using normal human umbilical vein endothelial cells (HUVEC) and normal human aortic smooth muscle cells (HAoSMC) on both SF and SF + REDV films to evaluate the impact of REDV on endothelialization. The results indicated a trend towards promoting HUVEC proliferation while inhibiting HAoSMC proliferation. Therefore, these findings suggest that SF + REDV may be more suitable than SF alone for coating small-diameter SF knitted tubes made of SF threads.

Multifunctional nanoparticle-VEGF modification for tissue-engineered vascular graft to promote sustained anti-thrombosis and rapid endothelialization.

Purpose: The absence of a complete endothelial cell layer is a well-recognized reason leading to small-diameter tissue-engineered vascular graft failure. Here we reported a multifunctional system consisting of chitosan (CS), Arg-Glu-Asp-Val (REDV) peptide, heparin, and vascular endothelial growth factor (VEGF) to achieve sustained anti-thrombosis and rapid endothelialization for decellularized and photo-oxidized bovine internal mammary arteries (DP-BIMA). Methods: CS-REDV copolymers were synthesized via a transglutaminase (TGase) catalyzed reaction. CS-REDV-Hep nanoparticles were formed by electrostatic self-assembly and loaded on the DP-BIMA. The quantification of released heparin and vascular endothelial growth factor was detected. Hemolysis rate, platelets adhesion, endothelial cell (EC) adhesion and proliferation, and MTT assay were performed in vitro. The grafts were then tested in a rabbit abdominal aorta interposition model for 3 months. The patency rates were calculated and the ECs regeneration was investigated by immunofluorescence staining of CD31, CD144, and eNOS antibodies. Results: The nanoparticle-VEGF system (particle size: 61.8 ± 18.3 nm, zeta-potential: +13.2 mV, PDI: .108) showed a sustained and controlled release of heparin and VEGF for as long as 1 month and exhibited good biocompatibility, a lower affinity for platelets, and a higher affinity for ECs in vitro. The nanoparticle-VEGF immobilized BIMA achieved 100% and 83.3% patency in a rabbit abdominal interposition model during 1 and 3 months, respectively, without any thrombogenicity and showed CD31, CD144, eNOS positive cell adhesion as early as 1 day. After 3 months, CD31, CD144, and eNOS positive cells covered almost the whole luminal surface of the grafts. Conclusion: The results demonstrated that the multifunctional nanoparticle-VEGF system can enhance the anti-thrombosis property and promote rapid endothelialization of small-diameter tissue-engineered vascular grafts. Utilizing nanoparticles to combine different kinds of biomolecules is an appropriate technology to improve the long-term patency of small-diameter tissue-engineered vascular grafts.

A nitric oxide-eluting and REDV peptide-conjugated coating promotes vascular healing.

Drug-eluting stents (DESs) placement remarkably reduces the over-proliferation of smooth muscle cells (SMCs) and thus neointimal hyperplasia. However, the pharmacological agent also slows down the re-endothelization, delays injury vascular healing and increases the risk of in-stent restenosis (ISR). Here, inspired by mussel foot proteins (Mfps), a mimicking endothelium functional stent coating was efficiently fabricated by thiol-ene "click" reaction, consisting of catechol grafted chitosan (CS-C), zinc sulfate, and Arg-Glu-Asp-Val (REDV) peptide. The mimicking endothelium coating could continuously catalyze endogenous nitric oxide (NO) gas and maintain the bioactivity of REDV peptide. Compared with bare stents, the mimicking coatings significantly inhibited the acute thrombosis for the first 1-week, accelerated re-endothelization and decreased in-stent restenosis for 1- and 3-month after implantation. In addition, the synergistic effect of NO and REDV peptide also regulated inflammation response and promoted the expression of muscle fiber.

Covalent functionalization of decellularized tissues accelerates endothelialization.

In the field of tissue regeneration, the lack of a stable endothelial lining may affect the hemocompatibility of both synthetic and biological replacements. These drawbacks might be prevented by specific biomaterial functionalization to induce selective endothelial cell (EC) adhesion. Decellularized bovine pericardia and porcine aortas were selectively functionalized with a REDV tetrapeptide at 10-5 M and 10-6 M working concentrations. The scaffold-bound peptide was quantified and REDV potential EC adhesion enhancement was evaluated in vitro by static seeding of human umbilical vein ECs. The viable cells and MTS production were statistically higher in functionalized tissues than in control. Scaffold histoarchitecture, geometrical features, and mechanical properties were unaffected by peptide anchoring. The selective immobilization of REDV was effective in accelerating ECs adhesion while promoting proliferation in functionalized decellularized tissues intended for blood-contacting applications.

Zwitterionic hydrogel-coated heart valves with improved endothelialization and anti-calcification properties.

Valve replacement surgery is the golden standard for end-stage valvular disease due to the lack of self-repair ability. Currently, bioprosthetic heart valves (BHVs) crosslinked by glutaraldehyde (GA) have been the most popular choice in clinic, especially after the emerge of transcatheter aortic valve replacement (TAVR). Nevertheless, the lifespan of BHVs is limited due to severe calcification and deterioration. In this study, to improve the anti-calcification property of BHVs, decellularized heart valves were modified by methacrylic anhydride to introduce double bonds (MADHVs), and a hybrid hydrogel made of sulfobetaine methacrylate (SBMA) and methacrylated hyaluronic acid (MAHA) was then coated onto the surface of MADHVs. Followed by grafting of Arg-Glu-Asp-Val (REDV), an endothelial cell-affinity peptide, the BHVs with improved affinity to endothelial cell (SMHVs-REDV) was obtained. SMHVs-REDV exhibited excellent collagen stability, reliable mechanical property and superior hemocompatibility. Moreover, enhanced biocompatibility and endothelialization potential compared with GA-crosslinked BHVs were achieved. After subcutaneous implantation for 30 days, SMHVs-REDV showed significantly reduced immune response and calcification compared with GA-crosslinked BHVs. Overall, simultaneous endothelialization and anti-calcification can be realized by this strategy, which was supposed to be benefit for improving the main drawbacks for available commercial BHVs products.

Ulvan mediated VE cadherin antibody and REDV peptide co-modification to improve endothelialization potential of bioprosthetic heart valves.

An aging population and a rapid increase in the incidence of degenerative valve diseases have led to greater use of bioprosthetic heart valves (BHVs). The durability of glutaraldehyde cross-linked bioprostheses currently available for clinical use is poor due to calcification, coagulation, and degradation. Decellularization can partially reduce calcification by removal of xenogenic cells, but can also lead to thrombosis, which can be addressed by further surface modification. The natural sulfated polysaccharide ulvan possesses antithrombotic and anti-inflammatory properties, and can behave as a heparinoid to immobilize proteins through their heparin binding sites. VE-cadherin antibody and the Arg-Glu-Asp-Val (REDV) peptide can facilitate selective endothelial cell attachment, adhesion and proliferation. In this study, we functionalized decellularized porcine pericardium (DPP) with ulvan, REDV, and VE-cadherin antibody (U-R-VE). Ulvan was covalently modified to act as a protective coating and spacer for VE-cadherin antibody, and to immobilize REDV. In in vitro tests, we found that functionalization significantly and selectively promoted adhesion and growth of endothelial cells while reducing platelet adhesion, inflammation, and in vitro calcification of DPPs. In an in vivo subdermal implantation model, U-R-VE modified DPP exhibited greater endothelialization potential and biocompatibility compared with unmodified pericardium. Thus, U-R-VE modification provides a promising solution to the problem of preparing BHVs with enhanced endothelialization potential.

Identification of circulating cells interacted with integrin α4ß1 ligand peptides REDV or HGGVRLY.

Recently, artificial blood vessels modified by integrin α4ß1 ligand, such as REDV, showed endothelialization improvement and antithrombotic properties have been reported. Early endothelialization was affected by the type of circulating cells captured by the peptide in the initial transplantation state, however, it is still not clarified. In this study, we identified in vitro circulating cells bound with the peptides arginine-glutamic acid-aspartic acid-valine (REDV) or histidine-glycine-glycine-valine-arginine-leucine-tyrosine (HGGVRLY). The effect of free C- or N-terminal of HGGVRLY on the type of peptide-binding cells was also studied. The rat circulating cells were isolated from blood and incubated with 5(6)-carboxyfluorescein (5/6-FAM, F) labeled F-REDV (C-terminal free), F-HGGVRLY (C-terminal free), or HGGVRLY-F (N-terminal free). Furthermore, peptide-binding cells were identified by co-staining with various antibodies labeled with PE, PerCP/Cy5.5, or APC. N-terminal free HGGVRLY-F was found to bind to more circulating cells than C-terminal free F-REDV and F-HGGVRLY. The ratio of integrin α4ß1 positive cell bound with F-REDV, F-HGGVRLY, or HGGVRLY-F reached over 90 %, demonstrating that HGGVRLY is also a ligand of integrin α4ß1. Among identified cell types, we found that F-REDV mainly bounds with EPC and BMSC, while F-HGGVRLY with BMSC. HGGVRLY-F bounds with EPC and BMSC, exhibiting a higher EPC binding ratio than F-REDV and F-HGGVRLY.

Bioinspired, Artificial, Small-Diameter Vascular Grafts with Selective and Rapid Endothelialization Based on an Amniotic Membrane-Derived Hydrogel.

Clinical application of the amniotic membrane (AM) in vascular reconstruction was limited by poor processability, rapid biodegradation, and insufficient hemocompatibility. In this work, decellularized AM was digested to a thermosensitive hydrogel and densely cross-linked in the nanoscale as "enhanced" collagenous fibers. Via N-(3-dimehylaminopropyl)-N'-ethylcarbodiimide and N-hydroxysuccinimide (EDC/NHS) catalysis, REDV was further grafted to simulate anticoagulant substances on naturally derived blood vessels. This modification approach endowed AM with rapid endothelialization and rare vascular restenosis. Through adjusting the fixation condition, the pore size and mechanical stability of the fiber network were approximate to those of natural tissues and precisely designed to fit for cell adhesion. AM was synchronously fixed by alginate dialdehyde (ADA) and EDC/NHS, forming a "double-cross-linked" stable structure with significantly improved mechanical strength and resistance against enzymic degradation. The hemolytic and platelet adhesion test indicated that ADA/REDV-AM could inhibit hemolysis and coagulation. It also exhibited excellent cytocompatibility. It selectively accelerated adsorption and migration of endothelial cells (ECs) while impeding adhesion and proliferation of smooth muscle cells (SMCs). It maintained EC superiority in competitive growth and avoided thrombosis in vivo. Furthermore, its property of promoting reconstruction and repair of blood vessels was proved in an animal experiment. Overall, the present study demonstrates that ADA/REDV-AM has potential application as a small-diameter artificial vascular intima with rapid endothelialization and reduced SMC/platelet adhesion.

Arg-Glu-Asp-Val Peptide Immobilized on an Acellular Graft Surface Inhibits Platelet Adhesion and Fibrin Clot Deposition in a Peptide Density-Dependent Manner.

Acellular blood vessels possess high potential to be used as tissue-engineered vascular scaffolds. Previously, a high patency was achieved for an Arg-Glu-Asp-Val (REDV) peptide-immobilized small-diameter acellular graft in a minipig model. Results revealed the potential of the peptide to capture a circulating cell and also to suppress fibrin clot deposition. Here, the effect of REDV peptide density on the blood response under ex vivo blood perfusion conditions was investigated. When endothelial cells or platelets were seeded under static conditions, the number of adherent endothelial cells increased with the increase in peptide density. Platelets scarcely adhered on the surface where the peptide density was above 18.9 × 10-4 molecules per nm3. Fibrin clot deposition and circulating cell capture were evaluated in a minipig extracorporeal circulatory system. The fibrin clot did not form on the peptide-immobilized surface, in the range of peptide modification density that was evaluated, whereas the unmodified surface was covered with microthrombi. REDV-specific blood circulating cells were captured on the peptide-immobilized surface with a density above 18.9 × 10-4 molecules per nm3. These results illustrated, under ex vivo blood perfusion conditions, that the REDV-immobilized acellular surface was able to capture cells and also suppress platelet adhesion and fibrin clot deposition in a peptide density-dependent manner.

Phospholipid-based multifunctional coating via layer-by-layer self-assembly for biomedical applications.

As an important class of biomaterials，bionics inspired materials has been widely used in creating extracorporeal and implantable medical devices. However, specific service environment is often faced with multiple requirements rather than single function. Herein, we designed a phospholipid-based multifunctional coating with phospholipids-based polymers, type I collagen (Col-I) and Arg-Glu-Asp-Val (REDV) peptide, via layer-by-layer assembly. The successful synthesis of the polymers and the coating is proved by a series of characterization methods including Fourier transforming infrared spectra (FTIR), proton nuclear magnetic resonance (1H NMR), ultraviolet-visible spectra (UV) and X-ray photoelectron spectroscopy (XPS), while the assembly process and quality change of the coating were monitored via quartz crystal microbalance (QCM). Besides, hydrophilicity and roughness of this coating was analyzed via water contact angle (WCA) and atomic force microscope (AFM), respectively. Finally, results from platelet adhesion, activation assay, smooth muscle cells (SMCs) and endothelial cells (ECs) cultures indicated that the multifunctional coating could strongly inhibit platelet adhesion and SMCs proliferation, hence provide practical application of the coating with good biocompatibility, especially the anticoagulant property and cell compatibility. It is expected that this coating may be used in blood-contacting fields such as cardiovascular stent or other devices in the future.

An Efficient Surface Modification Strategy Improving Endothelialization with Polydopamine Nanoparticles and REDV Peptides for Stent-Grafts.

Stents or stent-grafts are often functionalized with films to enhance cell/surface interactions and improve endothelialization. However, continuous film coatings by common surface modification tactics may preclude cells from migrating along the thickness direction and may change the physical characteristics of stent-grafts. Here, polydopamine nanoparticles (PDA-NPs) are attached on braided stent-grafts tightly, forming a nanostructure on microfilaments. They also serve as the anchor for bioactive REDV peptide immobilization to promote endothelia cells (ECs) activities. The results show that braided stent-grafts decorated with PDA-NPs and REDV demonstrate an excellent endothelialization performance and hemocompatibility due to the micro/nanostructure formed and REDV affinity to ECs. The physical properties of stent-grafts are also not compromised. A potential surface modification strategy for scaffold applications is illustrated.

Multifunctional REDV-G-TAT-G-NLS-Cys peptide sequence conjugated gene carriers to enhance gene transfection efficiency in endothelial cells.

Rapid endothelialization on small diameter artificial blood vessels is an effective strategy to facilitate long-term patency and inhibit thrombosis. The gene delivery can enhance the proliferation and migration of endothelial cells (ECs), which is beneficial for rapid endothelialization. REDV-G-TAT-G-NLS-Cys (abbreviated as TP-G) peptide could weakly condense pEGFP-ZNF580 (pZNF580) and transfect ECs, but its transfection efficiency was still very low because of its low positive charge, low stability and weak endosome escape ability. In order to develop more stable and efficient gene carriers with low cytotoxicity, in the present study, we conjugated different amounts of TP-G peptide onto poly(lactide-co-glycolide)-g-polyethylenimine (PLGA-g-PEI) amphiphilic copolymers via a hetero-poly(ethylene glycol) spacer (OPSS-PEG-NHS). The TP-G peptide and PEI could cooperatively and strongly condense pZNF580. The carrier's cytotoxicity was reduced by the introduction of poly(ethylene glycol) spacer. They condensed pZNF580 to form gene complexes (PPP-TP-G/pZNF580) with suitable size and positive zeta potential for gene delivery. The transfected ECs promoted their migration ability as demonstrated by cell migration assay. The results of cellular uptake and confocal laser scanning microscopy showed significantly high internalization efficiency, endosomal/lysosomal escape and nucleus location of pZNF580 by this multifunctional TP-G peptide sequence conjugated gene delivery system. Furthermore, several inhibitors were used to study the cellular uptake pathways of PPP-TP-G/pZNF580 complexes. The results showed that PPP-TP-G2/Cy5-oligonucleotide complexes exhibited the optimized endocytosis pathways which facilitated for cellular uptake. In conclusion, the multifunctional TP-G peptide conjugated gene carriers could promote the transfection efficiency due to the multifunction of REDV, cell-penetrating peptide and nuclear localization signal in the peptide sequence, which could be a suitable gene carrier for endothelialization.

Peptide-Functionalized Polyurethane Coatings Prepared via Grafting-To Strategy to Selectively Promote Endothelialization.

Endothelialization, formation of endothelial cells (ECs) layer on cardiovascular implant surface, is considered an ideal approach to prevent restenosis (renarrowing of blood vessel mainly due to the accumulation of proliferated vascular smooth muscle cells, SMCs) and thrombosis. In this study, the possibility of using polyurethane (PU) as a coating platform for functionalization with peptide to enhance endothelialization on implants is explored. PUs are synthesized through metal-free organocatalytic polymerization followed by chemical conjugation with an EC-specific REDV peptide through thiol-ene reaction. Meanwhile, the free isocyanate groups of PU allow for covalent grafting of REDV-functionalized PU (PU/REDV) to silanize implant materials (nitinol and PET). PU/REDV coating with peptide grafting density of ≈2 nmol cm-2 selectively accommodates primary human umbilical vein ECs (HUVECs) and retards spreading of primary human umbilical artery SMCs (HUASMCs). In addition, a layer of HUVECs is formed within 3 d on PU/REDV-coated surfaces, while proliferation of HUASMCs is inhibited. The selectivity is further confirmed by coculture of HUVECs and HUASMCs. Moreover, the PU/REDV-coated surfaces are less thrombogenic as evidenced by reduced number and activity of adhered platelets. Therefore, PU/REDV can be potentially used as a coating of cardiovascular implants to prevent restenosis and thrombosis by promoting endothelialization.

Facile incorporation of REDV into porous silk fibroin scaffolds for enhancing vascularization of thick tissues.

Rapid neovascularization within scaffolds is critical for the regeneration of thick complex tissues. The surface immobilization of peptides and other active molecules have been explored to improve the vascularization capacity of implants. However, the rapid degradation of these molecules, the reaction conditions and cross-linking usually result in decreased vascularization capability. Here, we introduced a temperate, all-aqueous process to achieve bulk porous silk fibroin (SF) scaffolds. A temperature controlled process was used to induce the water stable structure by SF self-assembly. Arg-Glu-Asp-Val (REDV) peptides were added into SF solution and fixed within SF scaffolds during the self-assembly process. The results showed that the functionalized scaffolds markedly promoted the adhesion of endothelial cells in vitro. Moreover, the in vivo studies indicated enhanced cell infiltration in the bulk functionalized SF scaffolds and impressive vascularization at 4 weeks post-implantation. The functionalized scaffolds demonstrated excellent vascularization capability, providing an exciting biomaterial option for thick tissue regeneration.

Improving functional re-endothelialization of acellular liver scaffold using REDV cell-binding domain.

Engineering of functional vascularized liver tissues holds great promise in addressing donor organ shortage for transplantation. Whole organ decellularization is a cell removal method that retains the native vascular structures of the organ such that it can be anastomosed with the recipient circulation after recellularization with healthy cells. However, a main hurdle to successful implantation of bioengineered organ is the inability to efficiently re-endothelialize the vasculature with a functional endothelium, resulting in blood clotting which is the primary cause of failure in early transplant studies. Here, we present an efficient approach for enhancing re-endothelialization of decellularized rat liver scaffolds by conjugating the REDV cell-binding domain to improve attachment of endothelial cells (EC) on vascular wall surfaces. In order to facilitate expression and purification of the peptide, REDV was fused with elastin-like peptide (ELP) that confers thermally triggered aggregation behavior to the fusion protein. After validating the adhesive properties of the REDV-ELP peptide, we covalently coupled REDV-ELP to the blood vasculature of decellularized rat livers and seeded EC using perfusion of the portal vein. We showed that REDV-ELP increased cell attachment, spreading and proliferation of EC within the construct resulting in uniform endothelial lining of the scaffold vasculature. We further observed that REDV-ELP conjugation dramatically reduced platelet adhesion and activation. Altogether, our results demonstrate that this method allowed functional re-endothelialization of liver scaffold and show great potential toward the generation of functional bioengineered liver for long-term transplantation. STATEMENT OF SIGNIFICANCE: There is a critical need for novel organ replacement therapies as the grafts for transplantation fall short of demand. Recent advances in tissue engineering, through the use of decellularized scaffolds, have opened the possibility that engineered grafts could be used as substitutes for donor livers. However, successful implantation has been challenged by the inability to create a functional vasculature. Our research study reports a new strategy to increase efficiency of endothelialization by increasing the affinity of the vascular matrix for endothelial cells. We functionalized decellularized liver scaffold using elastin-like peptides grafted with REDV cell binding domain. We showed that REDV-ELP conjugation improve endothelial cell attachment and proliferation within the scaffold, demonstrating the feasibility of re-endothelializing a whole liver vasculature using our technique.