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Sternotomy as a Source of Autologous
Cells with Potential for Seeding

Aristotle D. Protopapas, Angela T. Riga,
Henryk J. Salacynski, Alexander M. Seifalian,
George Hamilton, Robin K. Walesby *

University Department of Surgery, Royal Free Hospital and
*Department of Cardiothoracic Surgery, University College Hospitals,
Royal Free and University College Hospitals, London, United Kingdom

SUMMARY
Introduction: The currently available synthetic conduits fail to achieve a patency equal to that of saphenous vein in cardiovascular bypass grafting. One-stage seeding with autologous mixed cells has been shown to improve their long-term patency for peripheral artery bypass in animal models. Mediastinal tissue could be utilised as an anatomically convenient source of cells for seeding in coronary bypass.
Objectives: The present experiment investigates enzymatic extraction of mixed cells from human mediastinal tissue within the anatomical and temporal constraints of one-stage seeding for coronary bypass surgery.
Material and Methods: Discardable anterior mediastinal tissue was harvested from 12 patients undergoing sternotomy for coronary surgery and subsequently cells were extracted utilising enzymatic harvesting. Cell count was performed. Timing of the processing was kept. Regression/correlation analysis was performed for weight/cell yield.
Results: The specimens yielded mixed-cell populations: endothelial, fibroblasts, erythrocytes and debris. The number of cells per gram of mediastinal tissue had a mean value of 0.6millions (range 0.02-1.3 millions), 95% confidence limits, 0.25-0.84 millions. Time required averaged 36 minutes. Multiple R was 0.7389916. Square R was 0.5461086.
Discussion: Cell extraction from human mediastinal tissue has succeeded in yielding a mixed-cell population with a positive correlation to the weight of the sample. Time required would permit one-stage seeding of conduits for coronary surgery.
Conclusion: Human mediastinal tissue may be utilized for further seeding experiments.

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BACKGROUND
    1. Procurement of an autologous conduit will always be an invasive procedure. Incising and dissecting into one anatomical site in order to treat pathology in another (even a physiologically more important indeed) area of the human body may test the medical and surgical principles of the Hippocratean'primum non nocere'.
   2. The currently available synthetic conduits fail to achieve a patency equal to that of saphenous vein in cardiovascular bypass grafting. The main reasons are:

a. Thrombogenic lumen: Neointima consisting of complete endothelial coverage was not present in Dacron or expanded polyterafluoroethylene grafts (1). Consequently, the normal platelet-endothelial cell interaction is missing, predisposing the grafts to acute thrombosis.
b. Neo-intimal hyperplasic: manifesting as sub-intimal fibroplasia, occurring at the heel, the toe of the anastomosis and the floor of the recipient artery. This was attributed to transformation of the pluripotent contractile arterial myoblast to proteinaceous-material-secretin- cell.
c. Haemodynamic mismatch: the lack of compliance (2) of synthetic conduits is creating turbulent flow and increased shear stress that have been held responsible for the transformation of the myoblast as above (b).

   3. One solution to the problems (a) and (b) is to create a neo-intima of infused autologous cells. For that, at least three methods have been described: seeding (3), sodding and pre-lining (4):

- Sodding is a procedure in which cells are infused prior to the bypass operation in two stages.
- Pre-lining is a complex procedure that involves multiple steps: harvesting by a preliminary operation, cell purification (5) and cell attachment. This would always be an expensive and time-consuming process requiring a specialized laboratory set-up and days of processing.
- Seeding entails infusing the cell suspension into the graft during the
revascularising procedure. This is a one-stage procedure. This last method is relatively simple and rapid.

OBJECTIVES
   By reflecting on experimental evidence, we have concluded that further exploration of one-stage mixed-cell seeding of appropriate synthetic conduits for coronary bypass was warranted. The standard median sternotomy for coronary bypass entails longitudinal division of the sternum and subsequent incision of the anterior mediastinal tissue plane before exposing the pericardial sac and the ascending aorta. In the adult human, this areolar plane consists of adipose tissue and elements of the regressed thymus gland. As this tissue may interfere with exposure and haemostasis, we excise and discard it. We have considered utilising this tissue for cell extraction. The cell yield could be seeded on synthetic conduits.


MATERIALS AND METHODS
The subjects were 12 patients undergoing coronary surgery with a median sternotomy.
   1. Surgical technique (Figure 1):
   During sternotomy for coronary surgery, the anterior mediastinal areolar tissue was sharply dissected and excised en bloc caudal to the innominate vein, cephalad to the diaphragm and anteriorly to the pericardial sac. Attention was given to sample as much tissue as possible to completely free the ascending aorta and the anterior pericardium. The surgical sample was placed in sterile universal 75ml- container with sodium chloride 0.9% and transferred immediately to the Tissue Culture Laboratory.

Figure 1

   2. Laboratory technique:
   The sample was weighed and then rinsed with Phoshate Buffer Solution (PBS) and minced with No 11 scalpel and the elastic end of the hub of a 5ml sterile disposable plastic syringe for injections (Plasti-Pad, B&T, and UK). The homogenisation of the suspension continued with repeated pipetting. It was then transferred to a 75-ml centrifugation tube containing 1500U/ml collagenase type II (Sigma UK) to a ratio: 1g/2ml. The tube was then incubated on a roller for 15 minutes at 37 degrees centigrade and 100 rounds per minute. The suspension was centrifuged at 1500/min for 10 minutes. The cell pellet was resuspended in culture medium (M199 of GIBCOBRL, supplemented with Hank's salts, 20% foetal calf serum, Penicillin 100IU/ml/Streptomycin 100μg/ml and 3.6 mM Glutamine). The new suspension was transferred to 25-ml culture flask (Falcon USA). Cell count was performed with a trypan blue assay and a manual haemocytometer. The flasks were incubated in 5% carbon dioxide/95% air at 37 degrees centigrade. Photo-microscopy was performed in 24 hours. Timing was kept between harvesting of the tissue specimen from the operating table and end of centrifugation. Relation between weight of specimen (explanatory variable) and cell yield (outcome variable) was sought by correlation analysis. Descriptive statistics and regression/correlation analysis were performed with Prism2 statistical package (GraphPad Software, Inc. San Diego, California USA).

RESULTS
   Specimen weight ranged between 3.1 and 22.7grams (median value 8, mean 6.1, and confidence level 95% 3.4). Specimens yielded mixed-cell populations on photo-microscopy: endothelial cells, fibroblasts, erythrocytes and debris. The number of viable cells per gram of mediastinal tissue had a mean value of 0.6 millions per gram. (Range 0.02-1.3 millions, 95% confidence limits, 0.25-0.84 millions). Time required ranged 30 to 40 minutes (median value 33.5, mean 34.6, and confidence level 95% 2). In performing regression/correlation analysis (explanatory variable, weight and outcome variable, cell yield) the following values were observed: Multiple R was 0.722805. Square R was 0.522447. Adjusted R square was 0.474691 and Standard Error 3.9

DISCUSSION
   Recent animal experiments have yielded encouraging results for single-stage seeding: Mixed-cell seeding of conventional Dacron (6) and expended polytetrafluoroethylene (7). The source of cells has varied widely: In the aforementioned animal experimental models cells have been harvested from autologous omental adipose tissue with relatively simple techniques. There have also been efforts with human mastectomy samples, which were successful, yet, time consuming (8). We attempt to apply the ideas and lessons learned from the above to the anatomico-physiological environment of coronary surgery.

   We have invesigated a method of extracting cells from within the operating field and within the standard time required for preparation for graft implantation. The anterior mediastinal tissue is anatomically appropriate for extracting cells for seeding of conduits in coronary surgery. The samples have yielded mixed cells with a simple processing. The time required would permit experimentation with one-stage seeding of 'off-the-shelf' conduits on the spot. There was a moderate correlation of the size (weight) of the harvested tissue and the tissue yield. This could be interpreted as a need to harvest as much as possible of the anterior mediastinal tissue on preparation in order to increase the yield. It is though unknown how this would affect the seeding process at a follow-up experiment.

CONCLUSION
   We have investigated whether extraction of mixed cells is possible from human mediastinal tissue in the time frame of a single-stage seeding regimen for coronary bypass surgery. This could bridge the previous animal and human experiments to one-stage seeding of conduits for coronary surgery.

ACKNOWLEDGEMENT
We are grateful to Messrs. Geoff Punshon and Graham Barden from the Tissue Culture Laboratory, Royal Free Hospital London UK for technical support.


REFERENCES

1. Sottiurai VS, Vidne BA, Lee AB Jr: Intimal hyperplasic and neointima: An ultrastractural analysis of thrombosed grafts in humans. Surgery 1983; 93: 809-817s.

2. Seifalian AM, Guidiceandrea A, Schmitz-Rixen T, Hamilton G: Non-compliance: the silent acceptance of a villain. From: Tissue Engineering of Vascular Grafts, Zilla P and Greisler HP (Eds.), RG Landes Co, Austin, Texas 1999 p.47-58

3.Herring M, Gardner A, Glover J: A single-stage technique for seeding vascular grafts with autogenous endothelium. Surg 1978; 77: 1382-7

4. Laube HR, Duwe J, Rutch W, Konertz W. Clinical experience with autologous endothelial cell-seeded polytetrafluoroethylene coronary artery bypass grafts. J Thorac Cardiovasc Surge 2000 Jul; 120(1): 134-41

5. Welch M, Durrans D, Carr HM, Vohra R, Rooney OB, Walker MG: Endothelial cell seeding: a review. J Vasc Surg 1992; 6(5): 473-84

6.Pasic M, Muller-Glauser W, von Segesser LK, Lachat M, Mihaljevic T, Turina MI: Superior late patency of small-diameter Dacron Grafts seeded with omental microvascular cells: an experimental study. Ann Thor Surg 1994; 58: 677-84

7. Frechette E, Dion YM, Cardon A Chakfe N, Doillon CJ: Fat- and Bone Marrow-impregnated small diameter PTFE grafts. Eur J Vasc Endovasc Surg 1999, 18: 308-314

8. Koyama M, Satoh K, Yoshida H, Suzuki S, Koie H, Takamatsu S: Surface coverage of vascular grafts with cultured human endothelial cells from subcutaneous fat tissue obtained with a biopsy needle. Thromb Haemost 1996 Oct: 76(4): 610-4

 

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2nd Virtual Congress of Cardiology

Dr. Florencio Garófalo
Steering Committee
President
Dr. Raúl Bretal
Scientific Committee
President
Dr. Armando Pacher
Technical Committee - CETIFAC
President
fgaro@fac.org.ar
fgaro@satlink.com
rbretal@fac.org.ar
rbretal@netverk.com.ar
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