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Biomedical subjects

J B Sharefkin

Publications and source records attributed to J B Sharefkin.

32 records · Page 2Linked to original sources
PubMed1986

Adult human endothelial cell enzymatic harvesting. Estimates of efficiency and comparison of crude and partially purified bacterial collagenase preparations by replicate microwell culture and fibronectin degradation measured by enzyme-linked immunosorbent assay.

Reliable enzymatic endothelial cell (EC) harvest methods are required for clinical EC seeding of vascular prostheses by methods analogous to those demonstrated in dogs. But crude collagenases used for EC harvest vary in efficacy and cytotoxicity, and purified collagenases reportedly give low EC yields. To compare different harvest methods, we studied growth curves of primary adult human saphenous vein EC (HSVEC) harvests plated in replicate microwell cultures. The EC yield, defined as attachment-capable ECs obtained per square centimeter of vein lumen, was estimated from the lowest number of ECs counted in lag phase before exponential growth began. With the use of morphometric studies of HSVs that were perfusion-fixed at their original dimensions, the baseline in situ density of ECs available for harvest from HSV was estimated at 1.3 X 10(5) EC/cm2. Crude (CBC) and partially purified bacterial collagenase (PBC) solutions at concentrations with equal levels of basement membrane lysis activity (BMLA) were compared by the replicate microwell method in a series of 21 harvests (six CBC, eight PBC, and seven enzyme-free control harvests). All 14 enzymatic harvests produced confluent EC cultures with no significant difference in mean harvest efficiency between CBC (12% of in situ EC number) and PBC (15%). However, PBC caused less degradation of human fibronectin (p less than 0.0001) as measured by an enzyme-linked immunosorbent assay employing a fibronectin-specific monoclonal antibody. These data suggest that chemically defined mixtures of pure enzymes with BMLA equal to the BMLA of crude collagenase might allow reliable EC harvesting without sacrifice in EC yield but with improved preservation of structures at the EC periphery. EC losses during initial vein dissection may have contributed to the low 12% to 15% efficiency we observed.

Adult↗
PubMed1986

Migration of adult human vascular endothelial cells: effect of extracellular matrix proteins.

An ideal vascular prosthetic graft should support rapid endothelial coverage and allow maximal rates of endothelial cell migration on its surface. To study this we modified and developed a method to measure rates of migration of adult human vascular endothelial cells (AHVECs) on test surfaces using an O-ring fence technique. We measured the rates of AHVEC migration on a variety of extracellular matrix proteins of interest because of their ability to support cell attachment. AHVECs, initially constrained in rings with an area of 0.62 cm2 (day 0), were allowed to migrate radially, and area coverage was measured after 7 days. The greatest areas of migration were seen on fibronectin (1.20 cm2) and gelatin (1.29 cm2) when compared with control (polystyrene [0.97 cm2]), p less than 0.01, p less than 0.05. Migration on laminin and prepared extracellular matrix from AHVEC was not statistically different from that of controls. Of extracellular matrix proteins studied in our system, a gelatin substrate in the presence of serum resulted in maximal rates of AHVEC migration.

Blood Vessel Prosthesis↗
PubMed1986

Use of tritiated thymidine as a marker to compare the effects of matrix proteins on adult human vascular endothelial cell attachment: implications for seeding of vascular prostheses.

We have developed a technique to measure attachment of adult human vascular endothelial cells to test surfaces with tritiated thymidine used as a marker. With this technique, we measured attachment of adult human vascular endothelial cells to a series of extracellular matrix proteins, including fibronectin-coated (10 micrograms/cm2), laminin-coated (10 micrograms/cm2), and collagen-coated (1% gelatin) surfaces because of the role of these proteins in promoting cell attachment and growth. For a typical experiment, in the presence of serum, initial attachment (at 1 hour) was greatest on fibronectin-coated (63%) and gelatin-coated (60%) tissue culture plastic (polystyrene) and was least on laminin-coated (28%) or untreated polystyrene (18%). The data suggest that fibronectin, either alone, or with a more complex combination of extracellular components may need to be present on prosthetic surfaces to produce maximal cell attachment and subsequent growth to confluence in vivo. The described method of measuring attachment is independent of surface properties, ensures complete recovery of cells, and will allow systematic exploration of those properties that best support human endothelial cell attachment to vascular prosthetic surfaces.

Blood Vessel Prosthesis↗
PubMed1984

Adult human saphenous vein endothelial cells: assessment of their reproductive capacity for use in endothelial seeding of vascular prostheses.

Autogenous endothelial seeding (AES) of vascular prostheses (VP) using venous endothelial cells (EC) reduces platelet-VP interactions and improves patency rates in small caliber VP in dogs. To conserve patients' veins for use in coronary or limb bypass surgery, human trials of AES should require proof that adequate numbers of EC with the growth capacity to cover VP can be harvested from acceptably small pieces of peripheral vein. EC were isolated from excess saphenous vein segments remaining after coronary bypass surgery by filling veins with 0.1% CLS II collagenase at 37 degrees C for 15 min and removing EC by flushing the veins with culture medium. EC were cultured on fibronectin-coated dishes in medium 199 with 30% human serum and 300 micrograms/ml of endothelial cell growth factor. These cells grew to form confluent monolayers, and were identified as EC by tests for factor VIII antigen. Veins from 53 patients with a mean age of 55.8 +/- 9.8 (SD) years yielded vein segments with an average area of 1.9 +/- 0.6 cm2, from which an average of 5.3 +/- 2.8 X 10(4) cells were removed per cm2 of vein area. EC in culture underwent 14.3 +/- 1.4 population doublings with an average population doubling time of 1.8 +/- 0.3 days (N = 14 cultures), which allowed an 100-fold increase in cell number to occur in 11 to 12 days. These data suggest that the EC available from small vein segments in adult humans have the growth capacity to cover areas comparable in size to the luminal areas of VP commonly used in arterial surgery.

Adult↗
PubMed1984

Platelet reactivity in vivo in dogs with arterial prostheses seeded with endothelial cells.

This study was designed to assess platelet activity in vivo with vascular prostheses seeded with endothelial cells to determine the time course for development of thromboresistance and to test the ability of prostheses to produce prostacyclin. Sixteen dogs were randomly allocated to receive seeded (experimental group) or unseeded (control group) velour Dacron aortic prostheses. Serial measurements of platelet survival were performed to assess platelet interaction with prostheses in vivo, and platelet serotonin was monitored as an index of platelet release in vivo. After placement of prostheses, dogs in the experimental group had rapid normalization of platelet survival, with most having normal platelet survival at 4 to 8 weeks after surgery. In contrast, most control animals had reduced platelet survival throughout the 12 week period of study. Significant differences between groups in mean platelet survival were noted at 8 weeks after surgery (p less than .005) and in mean platelet serotonin at 12 weeks after surgery (p less than .05). Luminal surface production of 6-keto-PGF1 alpha from seeded prostheses was similar to aortic production and significantly greater (p less than .05) than that of control prostheses. Gross thrombus was present on 6.0 +/- 3.4% of the prosthetic surface in experimental animals in comparison to 26.6 +/- 19.2% in controls (p less than .005). The results of these studies document accelerated nonreactivity with platelets of seeded prostheses due to rapid coverage with endothelium possessing a normal ability to produce prostacyclin.

Animals↗
PubMed1983

Endothelial cell labeling with indium-111-oxine as a marker of cell attachment to bioprosthetic surfaces.

Canine vascular endothelium labeled with indium-111-oxine was used as a marker of cell attachment to vascular prosthetic surfaces with complex textures. Primarily cultured and freshly harvested endothelial cells both took up the label rapidly. An average of 72% of a 32 micro Ci labeling dose was taken up by 1.5 X 10(6) cells in 10 min in serum-free medium. Over 95% of freshly labeled cells were viable by trypan blue tests and only 5% of the label was released after 1 h incubations at 37 degrees C. Labeled and unlabeled cells had similar rates of attachment to plastic dishes. Scanning electron microscopic studies showed that labeled cells retained their ability to spread on tissue culture dishes even at low (1%) serum levels. Labeled endothelial cells seeded onto Dacron or expanded polytetrafluoroethylene vascular prostheses by methods used in current surgical models could be identified by autoradiography of microscopic sections of the prostheses, and the efficiency of cell attachment to the prosthesis could be measured by gamma counting. Indium-111 labeling affords a simple and rapid way to measure initial cell attachment to, and distribution on, vascular prosthetic materials. The method could also allow measurement of early cell loss from a flow surface in vivo by using external gamma imaging.

Animals↗
PubMed1983

Seeding of Dacron vascular prostheses with endothelium of aortic origin.

Seeding of autologous venous endothelium on Dacron vascular prostheses in dogs results in endothelial coverage of the prosthetic flow surface 4-6 weeks after implantation. Canine aortic endothelium, in contrast, usually fails to completely cover an unseeded prosthesis by pannus ingrowth even over much longer periods. To see if the success of endothelial seeding stems from a difference in the ability of venous and aortic endothelium to grow on prosthetic surfaces, we seeded freshly harvested autologous aortic endothelium on Dacron velour infrarenal aortic prostheses in dogs. Six weeks after surgery these prostheses showed the features reported to be typical of seeded prostheses. Scanning electron micrographs showed a luminal lining of flat polygonal cells without fibrin or adherent formed blood elements, and light microscopy showed an underlying layer containing aligned spindle-shaped cells with elongated nuclei and cell-lined subluminal channels. Control prostheses were covered with fibrin and platelet-rich thrombi everywhere except for limited pannus ingrowth at anastomotic sites. The results suggest that the success of autologous endothelial seeding cannot be ascribed to inherent differences in properties such as mitotic capacity or fibrinolysis between venous and aortic endothelium. The formation of complete endothelial linings by seeding must instead result from a more favorable condition for endothelial cell growth created by the cell harvesting or seeding process itself.

Animals↗
PubMed1982

Early normalization of platelet survival by endothelial seeding of Dacron arterial prostheses in dogs.

Current synthetic vascular prostheses do not acquire a complete lining of vascular endothelium in humans or dogs. Seeding of autogenous venous endothelium has bee reported to remedy this defect, but previous studies have been primarily morphologic. To see if venous endothelial seeding caused a measurable decrease in platelet-prosthetic interaction in vivo, serial 111In-labeled platelet survival studies were done in dogs with seeded (N = 8) and unseeded (n = 9) thoracoabdominal Dacron bypass prostheses.l Changes in platelet survival time (PST) were compared with endothelial coverage scores, which were determined by blinded survey of removed prostheses by scanning electron microscopy (SEM). The PSTs before surgery (+/- SD) were 5.02 +/- 0.79 days (seeded) and 5.31 +/- 0.99 days (control) (P = 0.51). Seven weeks after surgery the PSTs were 4.01 +/- 1.10 days (seeded) and 2.67 +/- 0.88 days (control) (P = 0.013). The subgroup of four dogs with seeded prostheses that had complete endothelial coverage showed restoration of the platelet survival pattern to that of the linear decay seen before surgery, SEM studies showed that this normalization of PST occurred despite the presence of small nidi of platelet adhesion on exposed Dacron fibers and irregularities of the endothelial pattern consistent with flow disturbances and venous jet lesions near anastomoses. Endothelial seeding, when technically successful, appears to sharply curtail the degree of platelet interaction with vascular prostheses and restores a normal platelet survival pattern. Longer studies of seeded prostheses are needed to determine if seeding protects against the late complications of intimal hyperplasia and thrombotic occlusion.

Animals↗
PubMed1979

Successful management of early stroke after carotid endarterectomy.

This paper concerns the management of stroke coming on in the early postoperative period after successful carotid endarterectomy. Our experience in effectively reversing hemiplegia in three such consecutive patients forms the basis of this report. The value of instant reoperation is emphasized, and several factors that facilitate urgent reoperation have been identified. These include omission of preoperative angiography, immediate reexploration under local anesthesia, and rapid restoration of cerebral flow by insertion of a shunt. Our experience would indicate that reversal of neurological deficit in such patients can be accomplished if reoperation is carried out within one hour of onset of stroke. All three patients managed by these criteria recovered and were neurologically intact eight to twelve months later. These results are in contrast to the failure to reverse stroke noted by us and others when the above measures were not followed.

Aged↗