Int. J. Exp. Path. (1992), 73, 801-808
An immunohistochemical study of mesothelial cell seeding for knitted Dacron P.E. Bearn*, K. Millert, H. Bullt, A.M. Seddon§, C.N. McCollumT and A. Marston* *Bloomsbury Vascular Unit, The Middlesex Hospital, Mortimer Street, London WI 8AA; tDepartment of Histopathology, University College and Middlesex School of Medicine, University Street, London WC1 PE; Departments of fDermatology and §Histopathology, University College and Middlesex School of Medicine, Riding House Street, London WI P 7PN; and ¶Department of Surgery, University Hospital of South Manchester, Manchester M20 8LR, UK
Received for publication 19 March 1992 Accepted for publication 14 August 1992
Summary. Six greyhounds underwent bilateral femoral artery replacement with knitted Dacron, one side seeded with omental digest at graft preclotting, the other acting as an unseeded control. Grafts were removed at 24 hours and two months. Tissue was examined using a monoclonal antibody (MNF1 16) directed against a broad range of human cytokeratins to differentiate mesothelial cells (MC) from microvascular endothelial cells (MEC), which stained only with a polyclonal antibody directed against von Willebrand Factor (anti-vWF). Cells released from omentum by collagenase stained with MNF1 16 and reacted poorly with anti-vWF. Identical cells were observed to be within the interstices of seeded but not control knitted Dacron. Few remained in seeded grafts (n = 2) removed at 24 hours and none at two months (n=4).
Keywords: arterial graft, prosthesis, mesothelium, immunocytology Mesothelial cells (MC) from the peritoneal cavity form a non-thrombogenic surface in artificial circulations and proliferate in tissue culture (Clarke et al. 1984a; Nicholson et al. 1984). They are an alternative to endothelial cells (EC) for seeding on to prosthetic grafts where they have promoted luminal cell cover, increased antithrombotic activity and inhibited platelet deposition (Clarke et al. 1984b; Bull et al. 1988). MC may be obtained by exposing omentum to collagenase but identification has been based only on morphological and ultra-
structural criteria (Clarke et al. 1984b). Other workers have suggested that microvascular endothelial cells (MEC) and not MC are released and suggested that MEC were released in greater number if omentum was fragmented prior to enzymic digestion (Jarrell et al. 1986; Clarke & Pittilo 1987; Williams & Jarrell 1987). The recognition that mesothelium expresses cytokeratins which are not expressed by endothelium and that only EC express von Willebrand Factor (vWF) prompted immunocytochemical study of the
Correspondence: P.E. Bearn, Bloomsbury Vascular Unit, The Middlesex Hospital, Mortimer Street, London WI 8AA, UK. 8oi
P.E. Bearn et al. 802 while AuA1 failed to stain any tissue and fragmentation/collagenase technique (Dilley acted as the negative control. et al. 1979; Wagner et a]. 1982; Moll et al. 1983). These investigators concluded that MC and not MEC were the predominant cell released (Visser et al. 1991). The purpose of Mesothelial cell harvesting, seeding and graft this study was to use immunocytology to implantation validate the original technique of mesothelial Cell harvesting, graft seeding and implancell seeding (MCS) and to investigate reten- tation were carried out in one stage under tion of seeded cells in knitted Dacron general anaesthesia. implanted in the canine femoral artery for 24 hours and two months. Anaesthesia Materials and methods After induction with 10-20 ml 0.5% methohexitone sodium (Brietal Sodium, Lilly & Co Mesothelial seeding was performed on one Basingstoke, Hants, UK) injected into the Ltd, side (randomized) of bilateral superficial foreleg vein, the dogs were intubated and femoral artery replacement by knitted Dacmaintained on 30% 02/N20/1% C3H6, brearon in six female greyhounds (Wt 2 5-30 kg). thing spontaneously. 500 mg cefuroxime (Zinacef, Glaxo Laboratories Ltd, Middlesex, Primary antibodies UK) was given at induction and 12 hours post-operatively. Blood loss was replaced Mesothelial cells on paraffin sections and with normal saline. Intravenous fluid was cytospins were identified using a murine infused until the animal was taking oral anti-human cytokeratin monoclonal, fluids, at about 12 hours. DAKO-CK, MNF1 16 (Dakopatts, Dako Ltd, UK). This antibody reacts with a variety of human epithelial tissue and recognizes an Mesothelial harvesting epitope expressed on a broad range of cytokeratins, including 17 and 18 which are A minilaparotomy was performed to obtain 20 g of omentum which was incubated in expressed by mesothelial cells (Moll et al. 0.3% collagenase at 37°C for 20 minutes 1983; Goddard et al. 1991). (Boehringer Mannheim Gmbh, Germany). Cytospins and paraffin sections were The resulting cell suspension was washed stained with AuAl (Unipath Ltd, UK), a three times by centrifugation and resusmurine anti-human monoclonal antibody in 5 ml phosphate-buffered saline. pended which reacts with tumours of epithelial origin and is used to distinguish carcinoma from mesothelioma in cases of pleural effu- Mesothelial seeding of knitted Dacron sion. The epitope with which it reacts is unknown but in human tissue it does not Knitted Dacron (Microvel, Meadox (UK) Medicals Ltd, Dunstable, Beds, UK), 6 cm stain mesothelial cells. Specimens were also stained with a rabbit long x 6 mm internal diameter, was preclotted in three stages of 10 ml venous blood anti-von Willebrand Factor polyclonal antibody (anti-vWF) to identify EC (gift of Dr D. followed by a further 10 ml containing 10000 units of heparin (Yates et al. 1978). Dodds, New York State Health Department, MC were incorporated during the first and Empire Plaza, Albany, New York). Antibodies were tested against control second stages by adding 2-ml aliquots of the sections of canine artery, small bowel and seeding inoculum of 8 ml venous blood. peritoneum. MNF1 6 stained mesothelium Control grafts were preclotted as described but without the addition of the seeding cells. and epithelium, anti-vWF stained canine EC,
An immunohistochemical study of mesothelial cell seeding for Dacron
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Removal of grafts At 24 hours, two animals were re-aneasthetized. After heparinization with 20 000 units of heparin, grafts were removed and the animal then killed by intravenous injection of sodium pentobarbitone (Expirol, Ceva Ltd, Herts, UK). Grafts were similarly removed from the remaining four animals at two months.
Immunohistochemistry The following samples were taken for immunohistochemistry: omentum (collagenase treated and undigested), freshly seeded and control preclotted Dacron, seeded and control grafts explanted at 24 hours and two months. Canine tissue (small bowel, peritoneum, artery) was stained to check the affinities of test antibodies. Specimens were fixed immediately in formal saline and embedded in paraffin wax. Grafts were sectioned transversly at the midgraft and longitudinally at the anastomoses. Adjacent sections were taken for haematoxylin and eosin staining and immunohistochemistry. Paraffin sections were placed in a 5 6°C oven for 12-18 hours, then dewaxed and rehydrated. Endogenous peroxidase activity was inhibited by incubating sections with 0.5% H202 in methanol for 10-15 minutes before washing in running water for 5 minutes. All specimens were trypsinized for
Fig. 1. Canine omentum. Mesothelial cells are stained by MNF116 (arrowed). LM. x 180.
Fig. 2. Canine omentum. Vascular endothelium is stained by anti-vWF (arrowed). LM. x 180.
Surgical implantation All animals underwent bilateral replacement of the superficial femoral artery with a seeded graft on one side and an unseeded control on the other. The superficial femoral artery was fully exposed along its length and the profunda femoris ligated to prevent collateral formation. The superficial femoral artery was divided and a 4-cm segment removed and replaced with the Dacron graft anastomosed end to end using 6/0 prolene.
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Fig. 3. Canine omentum. After exposure to collagenase, mesothelium (arrowed) is lifted from the underlying adipose tissue. In other sections it was absent. MNFI 16. LM. x 180.
5-10 minutes using freshly prepared enzyme, washed with cold tap water and then twice with tris buffered saline (TBS) for 5 minutes. Non-specific binding of protein was blocked by incubating sections with normal rabbit serum (Dakopatts, Dako Ltd, UK) diluted to 1/5 with TBS for 10 minutes. The primary antibody was then applied and after 30 minutes the slides were rinsed in TBS. Either biotinylated swine anti-rabbit serum or rabbit anti-mouse serum with normal human serum was applied for 30 minutes and the sections rinsed with TBS. Avidin-biotin complex horseradish peroxidase (Dakopatts, Dako Ltd, UK) was applied for 30 minutes and sections then treated with diaminobenzidine solution for 10 minutes. After washing with TBS and running water and counterstaining with Mayer's haematoxylin, the sections were dehydrated and mounted using Canada balsam and a glass cover slip. A positive result
demonstrated peroxidase brown (Guesdon et al. 1979). Immunocytology Aliquots of omental cells were resuspended in foetal calf serum (Gibco Ltd, UK) at a concentration of 106/ml. 80-pl aliquots were dispensed into plastic cuvettes and cytospun (400 r.p.m., two minutes) onto glass microscope slides (Cytospin II, Shandon Ltd, UK). These were fixed with acetone for 10 minutes, air dried and stained with the different monoclonal and polyclonal antibodies using the indirect alkaline phosphatase/anti-alkaline phosphatase (APAAP) technique. For APAAP staining, sections were washed with TBS and the primary murine monoclonal/polyclonal rabbit antibody applied for 30 minutes. After washing with TBS, the second or bridging antibody was applied for 30 minutes (rabbit anti-
An immunohistochemical study of mesothelial cell seeding for Dacron mouse for monoclonals and mouse antirabbit followed by rabbit anti-mouse for
805
polyclonals). Rewashing with TBS was followed by application of the label, APAAP, as the third layer. The bridging complex and enzymic label were reapplied twice. APAAP was developed using a fast red substrate with alkaline phosphatase developing red. Cytospins were also stained with Giemsa. Mesothelial cells are polygonal with an eccentric nucleus and cytoplasm which stains a distinctive blue with a shade of violet (Spriggs & Boddington 1989). Sections were examined blind by two independent observers.
Results Control and collagenase treated omentum Mesothelium on the surface of the omentum was stained by MNF1 16 but not by AuA1 or anti-vWF. Microvascular endothelium stained with anti-vWF but not with MNF1 16 or AuAl. In contrast, mesothelial cells were absent from much of the surface of the digested omentum but where present retained the staining pattern of undigested material (Figs 1-3).
Seeding inoculum Cytospin preparations of the seeding inoculum contained all blood cellular elements. However, there were also sheets of uniform cells with the cytological appearance of mesothelium. These stained positively with MNF1 16 but not AuA1. Identical cells also stained weakly with anti-vWF (Fig. 4). Freshly seeded and unseeded Dacron Freshly seeded Dacron differed markedly from unseeded preclotted grafts. In seeded grafts, there were clumps of nucleated cells within the interstitial and surface thrombus. These cells, which were absent from controls, stained positively with MNF1 16 but
Fig. 4. Cytospin of the cell suspension derived by exposure of canine omentum to collagenase. Mesothelial cells are stained by MNF1 16 LM. x240.
not with AuAl. Both seeded and unseeded grafts demonstrated positive staining with the anti-vWF polyclonal but this was associated with thrombus (Fig. 5).
Seeded and unseeded Dacron at 24 hours Histological examination of mesothelial seeded grafts exposed to blood flow for 24 hours demonstrated strands of cells within the interstices of the seeded grafts. These were absent from unseeded controls, stained positively with MNF1 16 but failed to react with AuAl or anti-vWF, the latter again associated with thrombus. MNF11 6 staining cells were present in fewer numbers than in the freshly seeded grafts and those within the luminal thrombus had disappeared. The graft interstices of both seeded and control grafts were characterized by an infiltrate of neutrophil polymorphs (Fig. 6).
P.E. Bearn et al.
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5. Freshly seeded Dacron stained with MNF] 16. Mesothelial cells (arrowed) are distributed throughout the graft interstices. LM x 180.
Fig.
Seeded and unseeded Dacron at two months In both seeded and control grafts, anastomotic incorporation was by pannus ingrowth from the host artery. This was covered by endothelium which stained positively with anti-vWF but failed to stain with MNF1 16 or AuAl. There were no cells staining with MNF1 16 within the graft interstices. In three seeded grafts and two unseeded grafts, there were multinucleated cells within the perianastomotic thrombus which stained with MNF1 16 but not anti-vWF or AuAl (Fig. 7). At the midgraft, the neointima was thin and less cellular than in the region of perianastomotic incorporation. There were no cells which stained positively with MNF1 16.
Discussion
This study demonstrates that mesothelial cells (MC) are removed from the surface of
omentum by collagenase digestion of nonfragmented omentum. Preclotting knitted Dacron with this, seeds the graft interstices with MC. After implantation in a model of small artery bypass, few remain within the graft interstices by 24 hours and none after two months. Identification of MC within the cell digest released from omentum by collagenase had been based on cell morphology in tissue culture and deduced from the absence of cytoplasmic Weibel-Palade bodies, characteristic of EC (Nicholson et al. 1984; Clarke et al. 1984b). In this study, MC identity was confirmed using immunocytochemistry to demonstrate the cytokeratin which is characteristic of this cell (Schlegel et al. 1980; LaRocca & Rheinwald 1984; Wu et al. 1982). The weak staining with anti-vWF was due either to coating with vWF released from transected omental vessels or to nonspecific staining, a recognized problem with polyclonal anti-vWF antibodies in the absence of monoclonals (Visser et al. 1991).
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An immunohistochemical study of mesothelial cell seeding for Dacron
Fig. 6. Mesothelial seeded graft removed from the canine femoral artery at 24 hours. Few mesothelial cells (arrowed) are still present within the graft interstices. MNF116. LM. x 180.
Omentum contains MEC and in other studies where omentum has been fragmented before collagenase digestion, polyclonal antibodies may have misled researchers in to believing MEC and not MC to have been released (Jarrell et al. 1986; Clarke & Pittilo 1987; Williams & Jarrell 1987). Visser has now demonstrated that even with this modification, mesothelial cells are the predominant cell type, both in number and proliferative potential (Visser et al. 1991). We demonstrated that preclotting knitted Dacron with blood mixed with MC leads to cell retention at graft implantation. The technique is therefore quite suitable for patients where cell harvesting, graft seeding and implantation should ideally be performed under a single anaesthetic (Clarke et al.
1984b).
807
Fig. 7. Mesothelial seeded graft removed from the canine femoral artery at two months. No mesothelial cells are present within the graft interstices. Only macrophages (arrowed) within the luminal thrombus contain MNF1 16 staining material. MNF116. LM. x 180.
It is disappointing therefore that seeded MC are not retained in knitted Dacron at 24 hours: the preclot had disappeared and few MC remained. Some cells may have been washed off, unable to resist the shear rate acting at the graft surface, as reported in studies ofendothelial cell seeding (Rosenman et al. 1985). However, the leucocytic infiltrate suggests that MC may have been the target of cell mediated and humoral cytotoxicity (Callow 1987). Since so few MC were present at 24 hours, failure to demonstrate MC on seeded grafts removed at two months was hardly surprising. The multinucleate cells stained by MNF1 16 were probably macrophages which had phagocytosed cytokeratin debris, since usually only plasma cells are stained with this monoclonal (Wotherspoon et al. 1989).
P.E. Bearn et al. L (1986) Use of freshly isolated capillarry In summary, immunohistology, while endothelial cells for the immediate establishconfirming the technique of MC harvesting of a monolayer on a vascular graft at ment and seeding as first described by Clarke, also surgery. Surgery 100, 392-399. suggests that MC retention after graft LAROCCA P.J. & RHEINWALD J.G. (1 984) Expression implantation is too low for confluent mesoof simple epithelial keratins and vimentin by thelial cell cover to develop. human mesothelium and mesthelioma in vivo
8o8
and in culture. Cancer Res. 44, 2991-2999.
Acknowledgements This study was supported by Meadox (UK) Ltd. PEB was a British Heart Foundation Junior Fellow during the period of this study. References BULL H.A., PITTILo R.M., DRURY J., POLLOCK J.G., CLARKE J.M.F., WOOLF N., MARSTON A. & MACHIN S. ( 1988) Effects of autologous mesothelial seeding on prostacyclin production with Dacron arterial prosthesis. Br. 1. Surg. 75, 671. CALLOW A.D. (1987) Endothelial cell seeding: Problems and expectations. l. Vasc. Surg. 6,
318-319. CLARKE J.M.F. & PITTILO R.M. (1987) Vascular graft seeding. Surgery 102, 890. CLARKE J.M.F., PITTILO R.M., MACHIN S.J., WOOLF N. & MARSTON A. (1984a) A study of the possible role of mesothelium as a surface for flowing blood. Thromb. Haemostas. 51, 57-60. CLARKE J.M.F., PITTILO R.M., NICHOLSON L.J., WOOLF N. & MARSTON A. (1984b) Seeding Dacron arterial prostheses with peritoneal mesothelial cells: a preliminary morphological study. Br. l. Surg. 71, 492-494. DILLEY R., HERRING M., BOXER L., GARDNER A. & GLOVER J. (1979) Immunofluorescent staining for factor VIII-related antigen: a tool for study of healing in vascular prostheses. J. Surg. Res. 2 7, 149-55. GODDARD M.J., WILSON B. & GRANT J.W. (1991) Comparison of commercially available cytokeratin antibodies in normal and neoplastic adult epithelial and non-epithelial tissues. I Clin. Pathol. 44, 660-663. GUESDON J.L., TERYNCK T. & AVRAMEAS S. (1979) The use of avidin-biotin interaction in immunoenzymatic techniques. 1. Histochem. Cytochem. 27, 1131-1139. JARRELL B.E., WILLIAMS S.K., STOKES G., HUBBARD F.A., CARABASI R.A., KOOLPE E., GREENER D., PRATT K., MORITZ M.J., RADAOMSKI J. & SPEICHER
MOLL R., LEVY R., CZERNOBILSKY B., HOHLWEGMAJERT P., DALLENBACH-HELLWEG & FRANKE W.W. (1983) Cytokeratins of the normal epithelia and some neoplasms of the female genital
tract. Lab. Invest. 49, 599-610. NICHOLSON L.J., CLARKE J.M.F., PITrILO R., MACHIN S.J. & WOOLF N. (1984) The mesothelial cell as a non-thrombogenic surface. Thromb. Haemostas.
52, 102-104. ROSENMAN J.F., KEMPCZINSKI R.F., PEARCE W.H. & SILBERSTEIN E.B. (1985) Kinetics of endothelial cell seeding. I. Vasc. Surg. 2, 778-784. SCHLEGEL R., BANKS-SCHLEGEL S & PINKUs G.S. (1980) Immunohistochemical localisation of keratin in normal human tissues. Lab. Invest. 42, 91-96. SPRIGGS A.I. & BODDINGTON M.M. (1989) Atlas of Serious Fluid Cytopathology: a guide to the cells of the pleural, pericardial, peritoneal, and hydrocoele fluids. Kluwer Academic Publishers, USA. VISSER J.T., VAN BOCKEL J.H., VAN MUIJEN G.N.P. & VAN HINSBERGH V.W.M. (1991) Cells derived from omental fat tissue and used for seeding vascular prostheses are not endothelial in origin. J. Vasc. Surg. 13, 3 73-381. WAGNER D.D., URBAN-PICKERING & MARDER V.J. (1982) Immunolocalisation of Von Willebrand protein in Weibel Palade bodies of human endothelial cells. J. Cell. Biol. 95, 355-360. WILLIAMS S.K. & JARRELL B.E. (1987) Vascular graft seeding-reply. Surgery 102, 890-891. WOTHERSPOON A.C., NORTON A.J. & ISAACSON P.G. (1989) Immunoreactive cytokeratins in plasmacytomas. Histopathology 14, 141-1 50. WU Y.J., PARKER L.M., BINDER N.E., BECKET M.A., SINARD J.H., GRIFFITHS C.T. & RHEINWALD J.G. (1982) The mesothelial keratins: a new family of cytoskeletal proteins identified in cultured mesothelial cells and non-keratinising epithelia. Cell 31, 693-703. YATES S.G., BARROS D'S.A., BERGER K., FERNANDEZ L.G., WOOD S.1., RITTENHOUSE E.A., DAVIS C.C., MANSFIELD P.B. & SAUVAGE L.R. (1978) The preclotting of porous arterial prosthesis. Ann. Surg. 188, 611-622.
An immunohistochemical study of mesothelial cell seeding for knitted Dacron P.E. Bearn*, K. Millert, H. Bullt, A.M. Seddon§, C.N. McCollumT and A. Marston* *Bloomsbury Vascular Unit, The Middlesex Hospital, Mortimer Street, London WI 8AA; tDepartment of Histopathology, University College and Middlesex School of Medicine, University Street, London WC1 PE; Departments of fDermatology and §Histopathology, University College and Middlesex School of Medicine, Riding House Street, London WI P 7PN; and ¶Department of Surgery, University Hospital of South Manchester, Manchester M20 8LR, UK
Received for publication 19 March 1992 Accepted for publication 14 August 1992
Summary. Six greyhounds underwent bilateral femoral artery replacement with knitted Dacron, one side seeded with omental digest at graft preclotting, the other acting as an unseeded control. Grafts were removed at 24 hours and two months. Tissue was examined using a monoclonal antibody (MNF1 16) directed against a broad range of human cytokeratins to differentiate mesothelial cells (MC) from microvascular endothelial cells (MEC), which stained only with a polyclonal antibody directed against von Willebrand Factor (anti-vWF). Cells released from omentum by collagenase stained with MNF1 16 and reacted poorly with anti-vWF. Identical cells were observed to be within the interstices of seeded but not control knitted Dacron. Few remained in seeded grafts (n = 2) removed at 24 hours and none at two months (n=4).
Keywords: arterial graft, prosthesis, mesothelium, immunocytology Mesothelial cells (MC) from the peritoneal cavity form a non-thrombogenic surface in artificial circulations and proliferate in tissue culture (Clarke et al. 1984a; Nicholson et al. 1984). They are an alternative to endothelial cells (EC) for seeding on to prosthetic grafts where they have promoted luminal cell cover, increased antithrombotic activity and inhibited platelet deposition (Clarke et al. 1984b; Bull et al. 1988). MC may be obtained by exposing omentum to collagenase but identification has been based only on morphological and ultra-
structural criteria (Clarke et al. 1984b). Other workers have suggested that microvascular endothelial cells (MEC) and not MC are released and suggested that MEC were released in greater number if omentum was fragmented prior to enzymic digestion (Jarrell et al. 1986; Clarke & Pittilo 1987; Williams & Jarrell 1987). The recognition that mesothelium expresses cytokeratins which are not expressed by endothelium and that only EC express von Willebrand Factor (vWF) prompted immunocytochemical study of the
Correspondence: P.E. Bearn, Bloomsbury Vascular Unit, The Middlesex Hospital, Mortimer Street, London WI 8AA, UK. 8oi
P.E. Bearn et al. 802 while AuA1 failed to stain any tissue and fragmentation/collagenase technique (Dilley acted as the negative control. et al. 1979; Wagner et a]. 1982; Moll et al. 1983). These investigators concluded that MC and not MEC were the predominant cell released (Visser et al. 1991). The purpose of Mesothelial cell harvesting, seeding and graft this study was to use immunocytology to implantation validate the original technique of mesothelial Cell harvesting, graft seeding and implancell seeding (MCS) and to investigate reten- tation were carried out in one stage under tion of seeded cells in knitted Dacron general anaesthesia. implanted in the canine femoral artery for 24 hours and two months. Anaesthesia Materials and methods After induction with 10-20 ml 0.5% methohexitone sodium (Brietal Sodium, Lilly & Co Mesothelial seeding was performed on one Basingstoke, Hants, UK) injected into the Ltd, side (randomized) of bilateral superficial foreleg vein, the dogs were intubated and femoral artery replacement by knitted Dacmaintained on 30% 02/N20/1% C3H6, brearon in six female greyhounds (Wt 2 5-30 kg). thing spontaneously. 500 mg cefuroxime (Zinacef, Glaxo Laboratories Ltd, Middlesex, Primary antibodies UK) was given at induction and 12 hours post-operatively. Blood loss was replaced Mesothelial cells on paraffin sections and with normal saline. Intravenous fluid was cytospins were identified using a murine infused until the animal was taking oral anti-human cytokeratin monoclonal, fluids, at about 12 hours. DAKO-CK, MNF1 16 (Dakopatts, Dako Ltd, UK). This antibody reacts with a variety of human epithelial tissue and recognizes an Mesothelial harvesting epitope expressed on a broad range of cytokeratins, including 17 and 18 which are A minilaparotomy was performed to obtain 20 g of omentum which was incubated in expressed by mesothelial cells (Moll et al. 0.3% collagenase at 37°C for 20 minutes 1983; Goddard et al. 1991). (Boehringer Mannheim Gmbh, Germany). Cytospins and paraffin sections were The resulting cell suspension was washed stained with AuAl (Unipath Ltd, UK), a three times by centrifugation and resusmurine anti-human monoclonal antibody in 5 ml phosphate-buffered saline. pended which reacts with tumours of epithelial origin and is used to distinguish carcinoma from mesothelioma in cases of pleural effu- Mesothelial seeding of knitted Dacron sion. The epitope with which it reacts is unknown but in human tissue it does not Knitted Dacron (Microvel, Meadox (UK) Medicals Ltd, Dunstable, Beds, UK), 6 cm stain mesothelial cells. Specimens were also stained with a rabbit long x 6 mm internal diameter, was preclotted in three stages of 10 ml venous blood anti-von Willebrand Factor polyclonal antibody (anti-vWF) to identify EC (gift of Dr D. followed by a further 10 ml containing 10000 units of heparin (Yates et al. 1978). Dodds, New York State Health Department, MC were incorporated during the first and Empire Plaza, Albany, New York). Antibodies were tested against control second stages by adding 2-ml aliquots of the sections of canine artery, small bowel and seeding inoculum of 8 ml venous blood. peritoneum. MNF1 6 stained mesothelium Control grafts were preclotted as described but without the addition of the seeding cells. and epithelium, anti-vWF stained canine EC,
An immunohistochemical study of mesothelial cell seeding for Dacron
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Removal of grafts At 24 hours, two animals were re-aneasthetized. After heparinization with 20 000 units of heparin, grafts were removed and the animal then killed by intravenous injection of sodium pentobarbitone (Expirol, Ceva Ltd, Herts, UK). Grafts were similarly removed from the remaining four animals at two months.
Immunohistochemistry The following samples were taken for immunohistochemistry: omentum (collagenase treated and undigested), freshly seeded and control preclotted Dacron, seeded and control grafts explanted at 24 hours and two months. Canine tissue (small bowel, peritoneum, artery) was stained to check the affinities of test antibodies. Specimens were fixed immediately in formal saline and embedded in paraffin wax. Grafts were sectioned transversly at the midgraft and longitudinally at the anastomoses. Adjacent sections were taken for haematoxylin and eosin staining and immunohistochemistry. Paraffin sections were placed in a 5 6°C oven for 12-18 hours, then dewaxed and rehydrated. Endogenous peroxidase activity was inhibited by incubating sections with 0.5% H202 in methanol for 10-15 minutes before washing in running water for 5 minutes. All specimens were trypsinized for
Fig. 1. Canine omentum. Mesothelial cells are stained by MNF116 (arrowed). LM. x 180.
Fig. 2. Canine omentum. Vascular endothelium is stained by anti-vWF (arrowed). LM. x 180.
Surgical implantation All animals underwent bilateral replacement of the superficial femoral artery with a seeded graft on one side and an unseeded control on the other. The superficial femoral artery was fully exposed along its length and the profunda femoris ligated to prevent collateral formation. The superficial femoral artery was divided and a 4-cm segment removed and replaced with the Dacron graft anastomosed end to end using 6/0 prolene.
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Fig. 3. Canine omentum. After exposure to collagenase, mesothelium (arrowed) is lifted from the underlying adipose tissue. In other sections it was absent. MNFI 16. LM. x 180.
5-10 minutes using freshly prepared enzyme, washed with cold tap water and then twice with tris buffered saline (TBS) for 5 minutes. Non-specific binding of protein was blocked by incubating sections with normal rabbit serum (Dakopatts, Dako Ltd, UK) diluted to 1/5 with TBS for 10 minutes. The primary antibody was then applied and after 30 minutes the slides were rinsed in TBS. Either biotinylated swine anti-rabbit serum or rabbit anti-mouse serum with normal human serum was applied for 30 minutes and the sections rinsed with TBS. Avidin-biotin complex horseradish peroxidase (Dakopatts, Dako Ltd, UK) was applied for 30 minutes and sections then treated with diaminobenzidine solution for 10 minutes. After washing with TBS and running water and counterstaining with Mayer's haematoxylin, the sections were dehydrated and mounted using Canada balsam and a glass cover slip. A positive result
demonstrated peroxidase brown (Guesdon et al. 1979). Immunocytology Aliquots of omental cells were resuspended in foetal calf serum (Gibco Ltd, UK) at a concentration of 106/ml. 80-pl aliquots were dispensed into plastic cuvettes and cytospun (400 r.p.m., two minutes) onto glass microscope slides (Cytospin II, Shandon Ltd, UK). These were fixed with acetone for 10 minutes, air dried and stained with the different monoclonal and polyclonal antibodies using the indirect alkaline phosphatase/anti-alkaline phosphatase (APAAP) technique. For APAAP staining, sections were washed with TBS and the primary murine monoclonal/polyclonal rabbit antibody applied for 30 minutes. After washing with TBS, the second or bridging antibody was applied for 30 minutes (rabbit anti-
An immunohistochemical study of mesothelial cell seeding for Dacron mouse for monoclonals and mouse antirabbit followed by rabbit anti-mouse for
805
polyclonals). Rewashing with TBS was followed by application of the label, APAAP, as the third layer. The bridging complex and enzymic label were reapplied twice. APAAP was developed using a fast red substrate with alkaline phosphatase developing red. Cytospins were also stained with Giemsa. Mesothelial cells are polygonal with an eccentric nucleus and cytoplasm which stains a distinctive blue with a shade of violet (Spriggs & Boddington 1989). Sections were examined blind by two independent observers.
Results Control and collagenase treated omentum Mesothelium on the surface of the omentum was stained by MNF1 16 but not by AuA1 or anti-vWF. Microvascular endothelium stained with anti-vWF but not with MNF1 16 or AuAl. In contrast, mesothelial cells were absent from much of the surface of the digested omentum but where present retained the staining pattern of undigested material (Figs 1-3).
Seeding inoculum Cytospin preparations of the seeding inoculum contained all blood cellular elements. However, there were also sheets of uniform cells with the cytological appearance of mesothelium. These stained positively with MNF1 16 but not AuA1. Identical cells also stained weakly with anti-vWF (Fig. 4). Freshly seeded and unseeded Dacron Freshly seeded Dacron differed markedly from unseeded preclotted grafts. In seeded grafts, there were clumps of nucleated cells within the interstitial and surface thrombus. These cells, which were absent from controls, stained positively with MNF1 16 but
Fig. 4. Cytospin of the cell suspension derived by exposure of canine omentum to collagenase. Mesothelial cells are stained by MNF1 16 LM. x240.
not with AuAl. Both seeded and unseeded grafts demonstrated positive staining with the anti-vWF polyclonal but this was associated with thrombus (Fig. 5).
Seeded and unseeded Dacron at 24 hours Histological examination of mesothelial seeded grafts exposed to blood flow for 24 hours demonstrated strands of cells within the interstices of the seeded grafts. These were absent from unseeded controls, stained positively with MNF1 16 but failed to react with AuAl or anti-vWF, the latter again associated with thrombus. MNF11 6 staining cells were present in fewer numbers than in the freshly seeded grafts and those within the luminal thrombus had disappeared. The graft interstices of both seeded and control grafts were characterized by an infiltrate of neutrophil polymorphs (Fig. 6).
P.E. Bearn et al.
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5. Freshly seeded Dacron stained with MNF] 16. Mesothelial cells (arrowed) are distributed throughout the graft interstices. LM x 180.
Fig.
Seeded and unseeded Dacron at two months In both seeded and control grafts, anastomotic incorporation was by pannus ingrowth from the host artery. This was covered by endothelium which stained positively with anti-vWF but failed to stain with MNF1 16 or AuAl. There were no cells staining with MNF1 16 within the graft interstices. In three seeded grafts and two unseeded grafts, there were multinucleated cells within the perianastomotic thrombus which stained with MNF1 16 but not anti-vWF or AuAl (Fig. 7). At the midgraft, the neointima was thin and less cellular than in the region of perianastomotic incorporation. There were no cells which stained positively with MNF1 16.
Discussion
This study demonstrates that mesothelial cells (MC) are removed from the surface of
omentum by collagenase digestion of nonfragmented omentum. Preclotting knitted Dacron with this, seeds the graft interstices with MC. After implantation in a model of small artery bypass, few remain within the graft interstices by 24 hours and none after two months. Identification of MC within the cell digest released from omentum by collagenase had been based on cell morphology in tissue culture and deduced from the absence of cytoplasmic Weibel-Palade bodies, characteristic of EC (Nicholson et al. 1984; Clarke et al. 1984b). In this study, MC identity was confirmed using immunocytochemistry to demonstrate the cytokeratin which is characteristic of this cell (Schlegel et al. 1980; LaRocca & Rheinwald 1984; Wu et al. 1982). The weak staining with anti-vWF was due either to coating with vWF released from transected omental vessels or to nonspecific staining, a recognized problem with polyclonal anti-vWF antibodies in the absence of monoclonals (Visser et al. 1991).
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An immunohistochemical study of mesothelial cell seeding for Dacron
Fig. 6. Mesothelial seeded graft removed from the canine femoral artery at 24 hours. Few mesothelial cells (arrowed) are still present within the graft interstices. MNF116. LM. x 180.
Omentum contains MEC and in other studies where omentum has been fragmented before collagenase digestion, polyclonal antibodies may have misled researchers in to believing MEC and not MC to have been released (Jarrell et al. 1986; Clarke & Pittilo 1987; Williams & Jarrell 1987). Visser has now demonstrated that even with this modification, mesothelial cells are the predominant cell type, both in number and proliferative potential (Visser et al. 1991). We demonstrated that preclotting knitted Dacron with blood mixed with MC leads to cell retention at graft implantation. The technique is therefore quite suitable for patients where cell harvesting, graft seeding and implantation should ideally be performed under a single anaesthetic (Clarke et al.
1984b).
807
Fig. 7. Mesothelial seeded graft removed from the canine femoral artery at two months. No mesothelial cells are present within the graft interstices. Only macrophages (arrowed) within the luminal thrombus contain MNF1 16 staining material. MNF116. LM. x 180.
It is disappointing therefore that seeded MC are not retained in knitted Dacron at 24 hours: the preclot had disappeared and few MC remained. Some cells may have been washed off, unable to resist the shear rate acting at the graft surface, as reported in studies ofendothelial cell seeding (Rosenman et al. 1985). However, the leucocytic infiltrate suggests that MC may have been the target of cell mediated and humoral cytotoxicity (Callow 1987). Since so few MC were present at 24 hours, failure to demonstrate MC on seeded grafts removed at two months was hardly surprising. The multinucleate cells stained by MNF1 16 were probably macrophages which had phagocytosed cytokeratin debris, since usually only plasma cells are stained with this monoclonal (Wotherspoon et al. 1989).
P.E. Bearn et al. L (1986) Use of freshly isolated capillarry In summary, immunohistology, while endothelial cells for the immediate establishconfirming the technique of MC harvesting of a monolayer on a vascular graft at ment and seeding as first described by Clarke, also surgery. Surgery 100, 392-399. suggests that MC retention after graft LAROCCA P.J. & RHEINWALD J.G. (1 984) Expression implantation is too low for confluent mesoof simple epithelial keratins and vimentin by thelial cell cover to develop. human mesothelium and mesthelioma in vivo
8o8
and in culture. Cancer Res. 44, 2991-2999.
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