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J Thorac Cardiovasc Surg 1994;108:1043-1048
© 1994 Mosby, Inc.

SURGERY FOR ACQUIRED HEART DISEASE

Rapid visualization of viable and nonviable endothelium on cardiovascular prosthetic surfaces by means of fluorescent dyes

C. Gillis, MDa, A. Haegerstrand, MD, PhDa, B. Ragnarson, MDa, L. Bengtsson, MD, PhDa,b


Stockholm, Sweden

Supported by the Swedish Heart & Lung Foundation, the Swedish Society for Medical Research, and the Swedish Society of Medicine, Konung Gustaf V:s and Drottning Victorias Stiftelse and the Swedish MRC (7126).

Received for publication Nov. 8, 1993. Accepted for publication June 2, 1994. Address for reprints: Caroline Gillis, MD, Karolinska Institute, Department of Neuroscience, Doktorsringen 17, S-171 77 Stockholm, Sweden.

Abstract

Increasing interest in endothelialization of synthetic and tissue cardiovascular prostheses in vitro emphasizes the need for simple and rapid methods to evaluate presence of endothelium on surfaces. Scanning electron microscopy is a commonly used method for this purpose. In this study we investigated alternative and more rapid staining methods. Human saphenous vein endothelial cells in culture and on cardiovascular prosthetic materials (pyrolytic carbon, cusps of bioprosthetic heart valves, pig aorta, and expanded polytetrafluoroethylene) were labeled by exposing them to medium containing 5-chloromethylfluorescein diacetate or 1,1-dioctadecyl-3,3,3',3'-tetramethylindo carbocyanine perchlorate. For comparison, specimens were also fixed and processed for scanning electron microscopy. A bright fluorescence of endothelial cells labeled with 5-chlormethylfluorescein diacetate or 1,1-deoctadecyl-3,3,3',3'-tetramethylindo carbocyanine perchlorate were clearly visualized in culture, on pyrolytic carbon, and on expanded polytetrafluoroethylene. Unfixed, prelabeled cells could be visualized immediately and unlabeled cells could be investigated for viability within 1 hour. Cells seeded on biologic tissue specimens could be visualized within 15 minutes with a modified hematoxylin-eosin staining. We suggest the use of these methods for rapid visualization of endothelium present on surfaces of cardiovascular prosthetic materials where they can partly replace the use of scanning electron microscopy. (J THORACCARDIOVASCSURG1994;108:1043-8)

Many attempts have been made to develop methods to obtain an autologous endothelial lining on cardiovascular prosthetic materials. The clinical results with intraoperative low-density endothelial seeding of synthetic vascular grafts in human beings have been relatively disappointing and the method has not been widely adopted. Go Go 1-3 The combination of little spontaneous ingrowth of human endothelial cells on the grafts and improvements of cell culture have raised the possibility of preoperative high-density endothelial seeding of cultured endothelial cells. A confluent endothelium can hereby be achieved before implantation. Go Go 4-6 A reduced thrombogenicity of in vitro endothelialized expanded polytetrafluoroethylene (ePTFE) vascular grafts implanted in human beings was recently reported. Go Go 7,8 In vitro endothelialization has also been applied to heart valve prostheses to reduce thrombogenicity, tissue inflammation, and degeneration by reestablishment of the natural barrier between prosthetic and blood components. Go Go 9,10 The feasibility of creating an autologous (recipient) endothelium with cultured human saphenous vein endothelial cells on viable and nonviable allografts or xenografts has been shown. Go 6 It has also been shown that human saphenous vein endothelial cells form a confluent monolayer on mechanical heart valve prostheses, Go 11 adhere to the Dacron sewing ring, and adhere to vascular prostheses covered with pyrolytic carbon. Go Go 11-13

At present the most commonly used method to view an endothelial surface is scanning electron microscopy. It can be applied only after fixation, dehydration, and gold coating and is thus time consuming. Furthermore, complicated fixation techniques are required to reduce artifacts on materials that do not shrink. Go 14 Light microscopy of routine or immunohistochemically stained cells requires fixation and sectioning. To transect synthetic cardiovascular prosthetic materials such as ePTFE, Dacron fabric, or pyrolytic carbon is furthermore difficult or impossible. Consequently, these methods do not permit close preoperative or postoperative evaluation or the use of a single endothelialized prosthesis for consecutive evaluation of attachment, growth, or detachment of the endothelial cells. A simple and rapid endothelial labeling method, enabling cell identification and consecutive, immediate, and if desired, sterile, inspection of living cells on untransectable surfaces could thus be desirable.

Recently, compounds that become fluorescent when metabolized intracellularly, for example, 5-chloromethylfluorescein diacetate (CMFDA), have made visualization of living cells in culture possible. The CMFDA compound is metabolized intracellularly in viable cells to a cell-impermeant and fluorescent state within 1 hour. By illumination of the dye-containing cells with an epifluorescence microscope setup the cells fluoresce. Go Go 15,16 We Go 17 have previously described a method for labeling of endothelial cells in culture or on biologic tissue specimens using an autofluorescent carbocyanine dye (1,1-dioctade cyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate, DiI), which is known to be distributed in the lipid-bilayer parts of both living or fixed cells. Go Go 18,19

In the present study the possibility of using CMFDA or DiI labeling to visualize human saphenous vein endothelial cells in culture and seeded on synthetic surfaces (ePTFE, pyrolytic carbon) and tissue surfaces (deendothelialized pig aorta and cusps of commercially available porcine heart valve bioprostheses) was examined. For the biologic tissue a modified hematoxylin-eosin staining was also used for visualization. The morphologic features were compared with surfaces examined by means of scanning electron microscopy.

MATERIALS AND METHODS

The use of human great saphenous veins was approved by the Ethics Committee at the Karolinska Hospital.

Cell culture and characterization
Endothelial cells from 3 to 5 cm residual segments of the great saphenous vein from patients undergoing coronary bypass surgery were enzymatically detached and cultured as described previously. Go 20 The human saphenous vein endothelial cells in culture were characterized by their cobblestone appearance, positive immunoreactivity for von Willebrand factor–related antigen, and prostacyclin production. Go 20

Preparation and endothelialization of prosthetic materials
The endothelialized surfaces of four different prosthetic materials were evaluated:

  1. Leaflets of mechanical heart valve prostheses (bileaflet model 700, kindly provided by Carbomedics, Inc., Austin, Tex.)
  2. Human serum or collagen precoated ePTFE vascular grafts (8 mm, kindly provided by Svenska Gore, Sweden) Go Go 6,21
  3. Isolated cusps of heart valve bioprostheses (Biocor, kindly provided by Biocor Industria, Belo Horizonte, Brazil), where the glutaraldehyde was neutralized in 0.8% L-glutamic acid solution Go Go Go 10,22,23
  4. Porcine aorta, mechanically deendothelialized and devitalized in sterile deionized water containing antibiotics Go 24 and divided into 1 x 1 cm specimens

The prosthetic specimens were placed in culture wells (4 cm2; 12-well plate, Costar Corp., Cambridge, Mass.) and endothelialization was performed by cell seeding at a cell density corresponding to 1.5 to 2 x 105 cells/cm2. Vascular ePTFE grafts were endothelialized during stepwise rotation as previously described. Go 6

Properties of CMFDA
The compound CMFDA (also called CellTracker Green, Molecular Probes Inc., Eugene, Ore.) belongs to a group of chloromethyl derivatives recently developed for labeling of living cells in vitro. Go Go 15,16

By the addition of CMFDA to the cell culture medium, the compound will freely permeate the cell membrane and cytosolic esterases will cleave off its acetates, producing a CMFDA derivative that is brightly fluorescent. Further, in a reaction thought to be mediated by glutathione S-transferase, Go 25 this CMFDA-derivative will conjugate to intracellular thiols and become cell-impermeant. The chloromethyl-glutathione derivative of CMFDA has the same fluorescent properties as that of fluorescein (FITC) and can be excited by either the 488 nm line of the argon laser or conventional epifluorescence equipped with FITC filter sets. Go 15

CMFDA labeling
The crystalline CMFDA was dissolved in water-free dimethylsulfoxide (Merck, Darmstadt, Germany), diluted 1:100, and stored at 4° C in the dark until use. For evaluation of sufficient dye concentration for labeling of the cells in culture, the CMFDA solution was added to minimal essential medium to final concentrations of 100, 75, 25, 12.5 or 6.25 µmol/L. After 30 minutes under culture conditions, the dye-containing medium was replaced with culture medium. The media were removed after another 30 minutes and cells in culture were inspected with or without previous fixation in a Nikon epifluorescence microscope (Nikon, Inc., Instrument Group, Melville, N.Y.) using FITC filters. Presence of labeled (CMFDA) cells was evaluated after five different procedures:

  1. Human saphenous vein endothelial cells were labeled in culture and examined immediately.
  2. Human saphenous vein endothelial cells labeled in culture were seeded on prosthetic materials and examined after 2 days.
  3. Unlabeled human saphenous vein endothelial cells were seeded on all cardiovascular prosthetic materials, kept for 2 to 7 days under culture conditions, and then labeled and immediately examined.
  4. The same procedure was used as in 3, but the cells were inspected after fixation in 4% paraformaldehyde in phosphate-buffered saline solution.
  5. The same procedure was used as in 3, but cells were fixed in 4% paraformaldehyde before labeling. This was done only on pyrolytic heart valve leaflets.

Labeled and unlabeled human saphenous vein endothelial cells on pyrolytic carbon leaflets were fixed in glutaraldehyde as described by Schroetter and colleagues Go 14 and further processed and evaluated in a Philips 515 scanning electron microscope (Philips Electronic Instruments, Inc., Mahwah, N.J.).

DiI labeling
The human saphenous vein endothelial cells in culture were labeled by the addition of DiI to the culture medium to a final concentration of 2.5 µg/ml for 2 days as described previously. Go 17 These cells were then detached and seeded on the prosthetic materials and kept under culture conditions for 2 to 7 days before examination, with or without previous fixation, in microscopes as described before but using Tetramethylrhodamine isothiocyanate filter setups (Leica AB, Kista, Sweden). Labeled specimens were stored in paraformaldehyde as described. DiI stains both viable and nonviable cells.

Modified hematoxylin-eosin staining
A slight modification of the standard hematoxylin-eosin staining technique Go 26 was used to stain fixed (4% paraformaldehyde, 5 minutes) endothelialized cusps of heart valve bioprostheses and reendothelialized porcine aorta. Specimens were stained in Mayer's hematoxylin-eosin stain for 6 minutes, rinsed in aqua fontana, left in lithium carbonate for 1 minute, rinsed twice in aqua fontana, stained in eosin for 2 minutes, rinsed in aqua fontana, rinsed in 95% alcohol, and finally stored in absolute alcohol. The specimens were examined in microscopes as described earlier. Specimens were stored in 99% ethanol.

RESULTS

In culture, the CMFDA concentration suitable for labeling was found to be 25 µmol/L and this concentration was used for all CMFDA labelings. CMFDA-labeled viable human saphenous vein endothelial cells in culture (Fig. 1, a), on ePTFE (Fig. 1, b) and on leaflets of pyrolytic carbon (Fig. 1, c) were clearly visualized in the epifluorescence microscope. On ePTFE, the endothelialization was easily distinguished as illustrated in the interface between human saphenous vein endothelial cells and a nonendothelialized area (Fig. 1, b). On collagen-precoated ePTFE grafts, the collagenous gel showed small weakly positive dots, which could be distinguished from positive cells by the lack of nuclei and by size. The CMFDA-labeled endothelium remained stained unfixed in dye-free culture medium when reevaluated 5 days after labeling. When the human saphenous vein endothelial cells on the respective prosthetic materials were fixed after labeling and stored in a dark environment, the cells were still clearly visible 2 months after labeling. The distinction of the endothelial cells was obvious, independent of whether the cells were labeled before seeding or in situ on the prosthetic materials. When the human saphenous vein endothelial cells were exposed to CMFDA after fixation, no stained cells were visualized (not shown).



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  		Fig. 1. Micrograph showing CMFDA-labeled human saphenous vein endothlial cells in culture (a) and seeded on an ePTFE graft and examined after 2 days (b). The edge is illustrating the difference between mechanically dendothelialized surface and intact, confluent endothelium. CMFDA-labeled human saphenous vein endothelial cells seeded on a leaflet of pyrolytic carbon are illustrated are illustrated in (c). For labeling, cells were incubated with CMFDA (25 µmol/L) for 30 minutes in medium without serum followed by at least 30 minutes in serum-containing culture medium before examining or seeding onto grafts. Scale bars denote 100 µm.

 
DiI-labeled endothelial cells on ePTFE or pyrolytic carbon were also clearly distinguishable with a brighter fluorescence than that of cells labeled with CMFDA, as illustrated on a leaflet made of pyrolytic carbon (Fig. 2, a). However, in favor of CMFDA staining for analysis of complete endothelialization, as well as for viability, the nature of the DiI-staining was more dotty and the edges of the cells were harder to distinguish. On the reendothelialized porcine aorta, the CMFDA- or DiI-labeled human saphenous vein endothelial cells were clearly distinguishable only at the edges of the porcine aorta specimens because of high background of light reflexes from the subendothelial matrix. On isolated cusps of biologic heart valve prostheses that had been chemically detoxified from glutaraldehyde, the cells were more easily distinguished in areas where the cusp was thin, that is, where light was not extensively reflected (not shown).



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  		Fig. 2. Micrograph illustrating DiI-labeled human saphenous vein endothelial cells seeded on pyrolytic carbon (a), hematoxylin-eosin stained cells seeded on pig aorta specimens (b), and scanning electron micrograph of CMFDA stained endothelial cells seeded on pyrolytic carbon (c). Cracks in the endothelium on the micrograph are due to preparation artifacts. For DiI labeling, human saphenous vein endothelial cells were incubated for 2 days with dye-containing (2.5 µg/ml) culture medium. Scale bars in a and b denote 100µm.

 
When the human saphenous vein endothelial cells on porcine aorta were labeled with the modified hematoxylin-eosin staining method, the cells were visualized with a prominent staining of the nuclei of the cells (Fig. 2, b) within 15 minutes. Areas of the biologic tissue that were not covered with endothelium became dark gray without stained nuclei, making a clear distinction possible.

The appearance of the cells corresponded well with the appearance in the scanning electron micrograph, as illustrated by CMFDA-labeled cells on pyrolytic carbon (Fig. 2, c). Cracks in the endothelium in the scanning electron micrograph are due to preparation artifacts.

The feasibility of using the different staining techniques on the prosthetic materials is summarized in GoTable I.


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  		Table I. Suggestions of when to use the respective staining methods.
 
Considerations for microscopic examination
With the normal epifluorescence microscope (Nikon), the surfaces could be viewed easily with low magnification objectives (4x), especially for endothelial cells in culture and on pyrolytic leaflets. However, for photographic illustrations on nonplanar motifs, for example, vascular ePTFE, pig aorta, or cusps from bioprostheses, the narrow focal plane limited the photographic quality although evaluation was still possible. Thus, for illustrations a better view was achieved when the specimens were flattened with a metal weight and examined through the glass in an inverted microscope (Leica Fluovert microscope, Leica AB, Kista, Sweden). Conventional fluorescence microscopes for nonplanar motifs can also be used by fixing the tissue between slides and applying light pressure.

DISCUSSION

In this study we describe different methods for rapid labeling and examination of endothelial cells using CMFDA, DiI, or a modified hematoxylin-eosin staining. Human saphenous vein endothelial cells in culture, seeded on ePTFE, pyrolytic carbon, and on biologic tissue, were clearly viewed by these methods.

Seeding of grafts with CMFDA- or DiI-prelabeled endothelial cells make almost immediate examination of attachment, spreading, and completeness of endothelialization possible. The CMFDA or DiI labeling, if performed after seeding, distinguishes cells, and neither labeling requires fixation or transection before viewing in an epifluorescence microscope. Furthermore, CMFDA can be used to distinguish only viable cells of various kinds. Inasmuch as cells labeled according to these methods were difficult to distinguish on thicker and thus light-reflecting bioprosthetic surfaces, for example, porcine aorta, we also report an alternative hematoxylin-eosin staining method for rapid cell visualization that does not require transection but does include fixation. Other techniques for visualization of fixed cells for similar purposes have been described previously (e.g., cresyl violet). Go 27 Although these techniques require a longer time before evaluation, they are possible to use. Autotransplantation of intraoperatively recruited or cultured endothelial cells has been suggested to improve the outcome of cardiovascular interventions such as prosthetic implantation (valves, vessels) or angioplasty procedures. The procedure involves a risk of failure because of poor cell adhesion, cell detachment, or cell malfunction because of altered phenotype, technical errors, or microbial contamination. Accumulating data emphasize the importance of endothelial viability and a high degree of confluence for cell retention and anticoagulant function at the time of blood flow restoration. For investigation of cell detachment of endothelialized vascular grafts in situ, radiolabeling methods, such as indium111 have been used, although their reliability hasbeen questioned. Go 28 These techniques are not useful, however, for evaluation of cell morphology. Recently, visualization of Lac-Z gene transduced endothelial cells on ePTFE grafts was demonstrated by means of a ß-galactosidase chromogenic substrate. Go 29 This technique requires fixation of the cells and further processing. With CMFDA or DiI labeling, a smaller segment or a control graft can rapidly be inspected to guarantee satisfactory endothelialization of viable endothelial cells immediately before implantation. A prosthesis with CMFDA- or DiI-labeled cells can also be inspected after implantation and the prosthesis further processed for scanning electron microscopy if desired. It can possibly also be used in computer-assisted image analysis systems to quantify the degree of endothelialization.

Although not investigated in the present study, the dyes are likely to label most other cell types in culture. When cells (e.g., epithelial cells, myoblasts, or chondrocytes) are transplanted to animals, these techniques may be used for detection of the implanted cells.

In conclusion, we describe different staining techniques for rapid visualization of endothelial cells on cardiovascular prosthetic tissues using fluorescent dyes. We suggest these compounds for use in cardiovascular research in experimental studies and for examination of endothelialized prostheses in clinical use.

Acknowledgments

We thank Miss A. Dennerman for expert laboratory work, Mrs. I. May for technical assistance with scanning electron microscopy, and Dr. M. Rydh-Rinder for discussion concerning the hematoxylin-eosin staining. We also thank Dr. A. Modin and Dr. J. Rinder at the Department of Pharmacology, Karolinska Institute, for providing pieces of porcine aorta.

Footnotes

From the Department of Neuroscience, a Karolinska Institute, and the Department of Thoracic Surgery, b Karolinska, Hospital, Stockholm, Sweden. Back

References

  1. Fasol R, Zilla P, Deutsch M, Grimm M, Fischlein T, Laufer G. Human endothelial cell seeding: evaluation of its effectiveness by platelet parameters after one year. J Vasc Surg 1989;9:432-6.[Medline]
  2. Örtenwall P, Wadenvik H, Kutti J, Risberg B. Endothelial cell seeding reduces thrombogenicity of Dacron grafts in humans. J Vasc Surg 1990;11:403-10.[Medline]
  3. Herring MB. The use of endothelial seeding of prosthetic arterial bypass grafts. Surg Annu 1991;2:157-71.
  4. Anderson JS, Price TM, Hanson SR, Harker LA. In vitro endothelialization of small-caliber vascular grafts. Surgery 1987;101:577-86.[Medline]
  5. Zilla P, Siedler S, Fasol R, Sharefkin JB. Reduced reproductive capacity of freshly harvested endothelial cells in smokers: A possible shortcoming in the success of seeding? J Vasc Surg 1989;10:143-8.[Medline]
  6. Bengtsson L, Rådegran K, Haegerstrand A. A new and simple technique to achieve a confluent and flow resistent endothelium on vascular ePTFE-grafts using human serum. Eur J Vasc Surg 1993-1994;8:182-7.
  7. Magometschnigg H, Kadletz M, Vodrazka M, et al. Prospective clinical study with in vitro endothelial lining of expanded polytetrafluoroethylene grafts in crural repeat reconstruction. J Vasc Surg 1992;15:527-35.[Medline]
  8. Zilla P, Deutsch M, Meinhart J, et al. Clinical in vitro endothelialization of femoropopliteal bypass grafts: an actuarial follow-up over three years. J Vasc Surg 1994;19:540-8.[Medline]
  9. Grabenwöger M, Grimm M, Eybl E, et al. Endothelial cell lining of bioiprosthetic heart valve material. J Card Surg 1992;7:79-84.[Medline]
  10. Bengtsson L, Rådegran K, Haegerstrand A. In vitro thelialization of commercially available heart valve bioprostheses with cultured human cells. Eur J Vasc Surg 1994;7:393-8.
  11. Bengtsson L, Haegerstrand A. Endothelialization of mechanical heart valves in vitro with cultured adult human cells. J Heart Valve Dis 1993;2:352-6.[Medline]
  12. Bernex F, Mazzucotelli JP, Roudiere JL, Benhaiem-Sigaux N, Leandri J, Loisance D. In vitro endothelialization of carbon-coated Dacron vascular grafts. Int J Artif Organs 1992;15:172-80.[Medline]
  13. Sbarbati R, Gianessi D, Cenni MC, Lazzerini G, Verni F, de Caterina R. Pyrolytic carbon coating enhances Teflon and Dacron fabric compatibility with endothelial cell growth. Int J Artif Organs 1991;14:491-8.[Medline]
  14. Schroetter D, Spiess E, Paweletz N, Benke R. A procedure for rupture-free preparation of confluently grown monolayer cells for scanning electron microscopy. J Electron Microsc Technol 1984;1:219-25.
  15. Poot M, Kavanagh TJ, Kang HC, Kang HC, Haugland RP, Rabinovitch PS. Flow cytometric analysis of cell cycle-dependent changes in cell thiol level by combining a new laser dye with Hoechst 33342. Cytometry 1991;12:184-7.[Medline]
  16. Zhang Y-Z, Olson N, Mao F, Roth B, Haugland R. New fluorescent probes for long-term tracing of living cells. FASEB J 1992;6:A1835.
  17. Ragnarson B, Bengtsson L, Haegerstrand A. Labeling with fluorescent carbocyanine dyes of cultured endothelial and smooth muscle cells by growth in dye-containing medium. Histochemistry 1992;97:329-33.[Medline]
  18. Honig MG, Hume RI. Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long term cultures. J Cell Biol 1986;103:171-87.[Abstract/Free Full Text]
  19. Honig MG, Hume RI. DiI and DiO: versatile fluorescent dyes for neuronal labeling and pathway tracing. Trans Int Neurol Soc 1989;12:333-42.
  20. Haegerstrand A, Gillis C, Bengtsson L. Serial cultivation of adult human endothelium from the great saphenous vein. J Vasc Surg 1992;16:280-5.[Medline]
  21. Haegerstrand A, Bengtsson L, Gillis C. Serum proteins provide a matrix for cultured endothelial cells on expanded polytetrafluoroethylene vascular grafts. Scand J Thorac Cardiovasc Surg 1993;27:21-6.[Medline]
  22. Gendler E, Gendler S, Nimni ME. Toxic reactions evoked by glutaraldehyde-fixed pericardium and cardiac valve tissue bioprosthesis. J Biomed Mater Res 1984;18:727-36.[Medline]
  23. Grimm M, Eybl E, Grabenwoger M, et al. Biocompatibility of aldehyde-fixed bovine pericardium: an in vitro and in vivo approach toward improvement of bioprosthetic heart valves. J THORAC CARDIOVASC SURG 1991;102:195-201.[Abstract]
  24. Bengtsson L, Ragnarson B, Haegerstrand H. Lining of viable and nonviable allogeneic and xenogeneic cardiovascular tissue with cultured adult human venous endothelium. J THORAC CARDIOVASC SURG 1993;106:434-43.[Abstract]
  25. Zhang Y-Z, Naleway JJ, Larison KD, Huang ZJ, Haugland RP. Detecting lacZ gene expression in living cells with new lipophilic, fluorogeic beta-galactosidase substrates. FASEB J 1991;5:3108-13.[Abstract]
  26. Bancroft JD, Cook CH. Manunal of histological techniques. Edinburgh: Churchill Livingstone, 1984:18-20.
  27. Miyata T, Conte MS, Trudell LA, Mason D, Whittermore AD, Birinyi LK. Delayed exposure to pulsatile shear stress improves cell retention of human saphenous vein endothelial cells on seeded ePTFE grafts. J Surg Res 1991;50:485-93.[Medline]
  28. Herring MB, Gunther GR, Etchberger KJ. 111-Indium is an unreliable in vivo label for vascular endothelial cells. Ann Vasc Surg 1991;5:424-8.[Medline]
  29. Newman KD, Nguyen N, Dichek DA. Quantification of vascular graft seeding by use of computer-assisted image analysis and genetically modified endothelial cells. J Vasc Surg 1991;14:140-6.[Medline]




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