Confluent Endothelial Lining Research Articles

Mussel-inspired triblock functional protein coating with endothelial cell selectivity for endothelialization

Surface modification of biomaterials for rapid endothelialization is a promising approach for improving long-term patency of artificial vascular grafts (e.g. polytetrafluoroethylene, PTFE) with small-caliber vascular (<6 mm). However, surfaces modified with traditional strategies using hydrophilic polymers may be excessively hydrophilic to limit endothelial cell adhesion and formation of confluent endothelial lining. In this study, a triblock functional protein cofp-MZY/R was fabricated with cell selectivity of endothelial cells (ECs) over smooth muscle cells (SMCs) for endothelialization on PTFE. This rational designed triblock protein consisted of mussel-inspired domain, zwitterionic polypeptide and bioactive peptides (YIGSR and REDV), in which Dopa was efficiently obtained with residue-specificity in vivo. The triblock protein could facilely form coating on PTFE surface and the resulting protein coating exhibited moderate nonspecific resistance of protein and platelets. Together with bioactive peptides tail, it was available for cell attachment on surfaces. As protein material, this coating displayed remarkable biocompatibility through cytotoxicity and hemolysis measurements. Moreover, cellular behavior assay demonstrated that triblock protein coating could selectively promote adhesion, proliferation and migration of ECs rather than SMCs. This mussel-inspired triblock functional protein coating indicated a promising strategy for endothelialization of artificial vascular grafts.

Shear‐stress preconditioning and tissue‐engineering‐based paradigms for generating arterial substitutes

In situ tissue engineering using shear-stress preconditioning and adhesive biomolecules is a new approach to autologous tissue engineering. In the present study, novel tissue-engineering grafts (TEGs) were preconditioned within an in vitro pulsatile flow circuit, with and without the addition of fibronectin (FN), to establish whether low-shear-stress conditions promoted endothelial cell (EC) retention and differentiation. TEGs ( n =24) were generated by the contraction and compaction of collagen(I) by porcine aortic smooth-muscle cells (SMCs) on to a compliant polyester graft scaffold. ECs were radiolabelled with [(111)In]indium tropolonate and seeded on to the luminal surface of the TEGs. Following organ culture in a bioreactor (7 days), TEGs were split into four groups ( n =six TEGs per group): Group A acted as controls with TEGs unmodified and seeded with radiolabelled ECs; Group B underwent luminal pre-coating with FN (75 microg/ml) prior to EC seeding; Group C underwent preconditioning within a pulsatile flow circuit at 10-20 microN (1-2 dyn)/cm(2) for 7 days prior to EC seeding, and Group D TEGs were preconditioned for 7 days at 1-2 dyn/cm(2), followed by luminal pre-coating with FN prior to EC seeding. The resistance to physiological shear stress of the seeded ECs was assessed using a gamma-radiation counter within a physiological flow circuit producing an arterial waveform with a mean shear stress of 93.2 microN (9.32 dyn)/cm(2). Environmental scanning electron microscopy (ESEM) was used to determine the distribution and degree of differentiation of the attached Ecs, and tissue-type-plasminogen-activator (tPA) assays provided a measure of function and viability. EC resistance to shear stress at 93.2 microN/cm(2) was significantly enhanced by a period of preconditioning (Group C) at 10-20 microN/cm(2), surface modification with FN (Group B), or both (Group D) when compared with control grafts (Group A). However, TEGs coated with FN whether preconditioned (Group D) or not (Group B) demonstrated the best results for EC retention. ESEM demonstrated near-confluent differentiated flattened ECs in both these cases. EC function was demonstrated by a steady increase in tPA production. Low-shear-stress preconditioning of TEGs enhances EC retention in vitro with an additional advantage demonstrated by pre-treatment with FN prior to endothelialization. These findings may be exploited in the development of tissue-engineered constructs to maintain a confluent endothelial lining.

Can collagen impregnated polyester arterial prostheses be recommended as small diameter blood conduits?

A collagen impregnated graft and its parent preclotted prosthesis were implanted as thoraco-abdominal bypasses in dogs for periods ranging from 4 hr to 6 months and evaluated for their ease of handling, imperviousness, and healing behavior in terms of luminal surface thrombogenicity using labeled platelets and fibrinogen, prostacyclin (PGI2) secretion, histomorphometric determination of internal capsule thickness, and histopathologic and scanning electron microscopic studies. The collagen impregnated graft was impervious to blood and both grafts showed excellent handling characteristics. Fibrin uptake was negligible on both grafts; however, platelet uptake was higher on the collagen impregnated graft than on the control graft at 4 and 24 hr. The healing behavior of the collagen impregnated graft was also found to be different than that of the control graft between 1 and 6 months post implantation. The development of a host collagenous internal capsule at the anastomoses, and a confluent endothelial lining, was observed in both grafts at 1 month; in later implantation periods, the healing of the medial region was found to be more irregular in the collagen impregnated grafts, showing a lower mean PGI2 secretion than the preclotted control grafts. Histomorphometric analysis showed the internal capsule on the collagen impregnated grafts to be thicker than on the control grafts for most periods of implantation. The current study illustrates that the healing process of collagen impregnated grafts is delayed and that bovine collagen has a stimulating effect on tissue encapsulation. Current impregnated polyester arterial prostheses therefore cannot be recommended as small diameter blood conduits.

Lining of viable and nonviable allogeneic and xenogeneic cardiovascular tissue with cultured adult human venous endothelium

With the aim of creating a confluent endothelial lining of cultured adult human saphenous vein endothelial cells on cardiovascular bioprosthetic tissues in vitro, we performed seeding on deendothelialized segments of viable or devitalized (in deionized water) human vein, porcine aorta, and bioprosthetic tissues preserved in glutaraldehyde. After being seeded, specimens were kept for 7 days under culture conditions. On glutaraldehyde-preserved tissue, seeding was performed after 3 weeks of elution of glutaraldehyde. Evaluation was performed with hematoxylin-eosin staining, immunohistochemical staining of von Willebrand's factor and of collagen IV-related antigens, and scanning and transmission electron microscopy. The origin of the cells as derived from culture was verified by vital staining with a carbocyanine dye. Evaluation revealed a confluent lining of cultured human saphenous vein endothelial cells similar to native endothelium on both viable and nonviable human and porcine tissues. Collagen IV-related immunoreactivity was demonstrated close to the endothelial cells, corresponding to a de novo-formed basement membrane. Organelles and a basement membrane were demonstrated by transmission electron microscopy. The human saphenous vein endothelial cells seeded on glutaraldehyde-preserved tissues showed initial adherence but rounded up and detached on the second day of culture, probably because of residual glutaraldehyde. This study demonstrates that the native endothelium of allogenic or xenogeneic viable and nonviable vascular tissue may be replaced by cultured endothelium in vitro. The structural similarities with a native endothelium suggest that in vitro endothelialization with cultured autologous endothelial cells may be used to improve performance of cardiovascular bioprostheses.

Comparison of immediate seeding of endothelial cells with culture lining of small diameter ePTFE carotid interposition grafts

This study is the first to compare chronic healing characteristics of immediately seeded grafts with those of grafts lined by autogenous venous endothelial cells in tissue culture prior to implantation. Ten mongrel dogs had a segment of external jugular vein excised for enzymatic harvest of endothelial cells. After approximately 21 days growth in tissue culture, 4 × 10 6 cells/ml were inoculated into a 6-cm length of 4 mm i.d. ePTFE for formation of a confluent lining in culture media. The remaining external jugular vein had its endothelial cells enzymatically harvested for immediate seeding of an identical length of preclotted ePTFE. Both grafts were implanted end-to-end in the carotid position and excised after 30 days. In 6 of the 10 dogs, grafts were patent bilaterally; all others were occluded. Planimetric measurements on patent grafts with immediate seeding showed a thrombus-free surface area of 56 ± 39% compared to 86 ± 15% for culture-lined grafts ( P = 0.046). Endothelial coverage was 70 ± 24% for immediately seeded grafts and 29 ± 21% for culture-lined grafts ( P = 0.016). We conclude that immediate seeding and culture lining of autogenous endothelial cells in small diameter ePTFE grafts produce equivalent short-term patency. While culture-lined grafts have an initially less thrombogenic luminal surface, subsequent development of a confluent endothelial lining is slower than that with an immediate seeding preparation, and thus would appear to offer no significant clinical benefit, especially in light of the complexity culture lining adds to the procedure.

Improved patency of collagen-impregnated grafts after in vitro autogenous endothelial cell seeding.

Currently available prosthetic vascular grafts remain sufficiently thrombogenic to preclude their use as small-caliber arterial substitutes. However, thrombogenicity may be significantly reduced by the presence of an endothelial monolayer on the luminal surface. The present study was undertaken to test the efficacy of lining a small-caliber prosthesis with autogenous endothelial cells in vitro so that the graft may subsequently be implanted with an established confluent endothelial lining. For this purpose, cells were obtained from canine external jugular vein, harvested enzymatically, and passaged in culture. Dacron grafts (4 X 150 mm) were then seeded in vitro and maintained for 48 to 72 hours before implantation in the femoral position of the same animal. Seeded grafts were implanted contralateral to unseeded control grafts and explanted after 1 month. Seeded grafts demonstrated an 86% patency rate at explanation in contrast to the significantly lower 14% patency rate of the unseeded control grafts. This study justifies further investigation directed toward the feasibility of endothelializing intravascular prostheses in vitro before implantation.

Improved patency of collagen-impregnated grafts after in vitro autogenous endothelial cell seeding

Currently available prosthetic vascular grafts remain sufficiently thrombogenic to preclude their use as small-caliber arterial substitutes. However, thrombogenicity may be significantly reduced by the presence of an endothelial monolayer on the luminal surface. The present study was undertaken to test the efficacy of lining a small-caliber prosthesis with autogenous endothelial cells in vitro so that the graft may subsequently be implanted with an established confluent endothelial lining. For this purpose, cells were obtained from canine external jugular vein, harvested enzymatically, and passaged in culture. Dacron grafts (4 × 150 mm) were then seeded in vitro and maintained for 48 to 72 hours before implantation in the femoral position of the same animal. Seeded grafts were implanted contralateral to unseeded control grafts and explanted after 1 month. Seeded grafts demonstrated an 86% patency rate at explanation in contrast to the significantly lower 14% patency rate of the unseeded control grafts. This study justifies further investigation directed toward the feasibility of endothelializing intravascular prostheses in vitro before implantation.

Endothelial cell seeding of a new PTFE vascular prosthesis

Previous attempts to line polytetrafluoroethylene (PTFE) prostheses with enzymatically derived endothelial cells have not been as successful as similar work with Dacron grafts because of the failure of such prostheses to develop a satisfactory subendothelium. This article reports our experience with a new, highly porous, unreinforced PTFE prosthesis that appears to circumvent this problem. Segments (4 mm I.D., 10 cm in length) of this new graft were implanted in 41 mongrel dogs as carotid interposition grafts. One graft in each dog was seeded with the dog's own endothelial cells, whereas the contralateral graft was treated in an identical fashion except for the inclusion of endothelium. After a mean period of 7 weeks of implantation, the grafts were harvested, their patencies were noted, the thrombus-free area of their luminal surface was calculated with computerized quantitative planimetry, and graft segments were submitted for scanning and transmission electron microscopy. In seven dogs the luminal surface was scraped from each graft and measured quantitatively. Although seeded grafts failed to show a statistically significant increase in patency during the short course of this experiment, a trend in that direction was quite striking. Furthermore, seeded grafts had a significant increase in thrombus-free area on their luminal surface as well as a significant reduction in the volume of luminal thrombus. Histologically, seeded grafts developed a substantial 75 to 100 μm cellular subendothelium beneath a confluent endothelial lining. No endothelial lining was noted in control grafts. We believe that the superior handling characteristics of this new prosthesis and its ability to develop a substantial subendothelium with a confluent endothelial lining suggest potential future applications and warrant further investigation.