Method for rapid adherence of endothelial cells onto a surface and surfaces prepared thereby

0001This invention relates to a method for attaching endothelial cells to a surface, and more specifically relates to a method for depositing a layer of endothelial cells on a polymeric surface and the surfaces prepared thereby.

0002Extensive investigations have been undertaken over many years to find materials that will be biologically and chemically stable toward body fluids. This area of research has become increasingly important with the development of various objects and articles which can be in contact with blood, such as artificial organs, vascular grafts, probes, cannulas, catheters and the like.

0003Synthetic plastics have come to the fore as preferred materials for such articles. However, such materials have the major drawback of being thrombogenic. Even such plastics as polytetrafluoroethylene and the silicone rubbers which are more compatible with blood than most plastics, still show thrombogenic characteristics.

0004Thrombogenicity has conventionally been counteracted by the use of anticoagulants such as heparin. Exemplary of heparinization procedures are the disclosures in U.S.-A-4,613,517 and U.S.-A-4,521,564.

0005Over the past three decades, artificial grafts have been used to restore blood flow to areas of ischemia, to provide blood flow for hemodialysis patients and for repair of arterial aneurysms. While these procedures are generally initially successful, long-term prognosis for patients receiving such grafts is not encouraging, principally because small diameter grafts (4mm or less) become occluded over time due to fibrin deposition and cellular adhesion due to the thrombogenic nature of the graft material.

0006The ideal blood-surface interface has long been considered to be the naturally occurring human endothelium, and much current research is focused on endothelialization procedures. Madri et al. in the <u style="single">Journal of Cell Biology</u> 97, 153 (1983) reported that when cells are grown on interstitial collagens, they undergo proliferation and form a continuous cell layer. Williams et al., <u style="single">Journal of Surgical Research</u> 38, 618 (1985) described pretreatment of prosthetic graft material with fibronectin, collagen or blood plasma, and reported that essentially no adherence occurred on untreated graft material, but that dramatic increases in adherence occurred on protein-coated polyester grafts. A similar study by Jarrell et al. (<u style="single">Annals of Surgery</u>, 203, 671 (1986)) showed a high percentage of firm adherence of endothelial cells to polyester coated with platelet-rich-plasma in 10 min., to amnion/collagen coated polyester in 30 min. and to plain polyester in two hours, but that only the amnion/collagen coated surface exhibited complete graft coverage.

0007In recent years, attention has focused upon the poor results generally obtained with small diameter vascular grafts. van Wachem et al., in <u style="single">Biomaterials</u> 6, 403 (1985) reported clinical success with polymeric grafts of greater than 4 mm, but that grafts of less than 4 mm gave generally disappointing clinical results due to immediate occlusion. Likewise, Baker et al., in <u style="single">American Journal of Surgery</u> 150, 197 (1985) stated that long term patency of large diameter vascular grafts is relatively acceptable, but small diameter (less than 4 mm) grafts exhibit poor long-term patency rates.

0008Seeding of 4 mm inside diameter polyester vascular grafts with endothelial cells and patency after implantation in dogs is discussed by Belden et al. in <u style="single">Trans. Am. Soc. Artif. Intern. Organs. 28</u>, 173 (1982).

0009Modification of polymeric surfaces by treatment with a variety of plasmas to achieve certain results is well known. For example, surface wettability, static properties and receptivity of a surface to deposition of a layer of an adherent polymeric material have been described. The doctoral thesis of Lee M. Smith, "Cell Adhesion As Influenced By Substrate Surface Properties", Department of Material Science and Engineering, the University of Utah, 1978, p. 67, suggests that cell adherence is a function of the carbon/oxygen ratio of the surface. van Wachem et al., (supra) discloses that endothelial cells can be cultured on glass or glow-discharge treated polystyrene.

0010U.S.-A-4,452,679 discloses a method to modify a polymeric surface to introduce specific chemical groups by treatment of the surface with a plasma in which at least one of the neutral, positive or negative species of the plasma is excluded from contacting the surface.

0011Endothelial cells adhere partially, but not confluently, to an untreated Dacron® polyester surface, that this surface will become confluently covered in 24 hours, and that near confluent coverage occurs with a Dacron® surface pretreated with a protein, such as platelet rich plasma. In the present disclosure, the term confluent is used to describe a surface which is substantially covered with cells which are contiguous in all directions.

0012EP-A-0 124 200 discloses the modification of polymeric surfaces by plasma treatment, whereby the plasma is generated from a gaseous material comprising, inter alia, nitrogen and/or oxygen.

0013The same is true for "Biomaterials" vol. 3, no. 2, April 1982.

0014From "Biomaterials", vol. 8, no. 5, September 1987 it is known that strong electrostatic interaction between the negatively charged cell-membrane and a positively charged surface occurs.

0015From Bruce E. Jarrell, Ann. Surg., June 1986, page 671 it is known that a confluent contact-inhibited monolayer of endothelial cells should have a density of 10⁵ endothelial cells per cm².

0016In spite of the extensive investigations on antithrombogenic prosthetic devices, the problem of thrombogenicity has not been satisfactorily solved, in particular with respect to small diameter grafts. It is toward the solution of this problem that the current invention is directed.

0017One aspect of the invention is a method to prepare a surface having a confluent layer of endothelial cells thereon according to claim 1. A polymeric substrate is exposed to a plasma generated from a material which includes nitrogen. The plasma treatment causes bonding of amino groups to the substrate. The substrate containing amino groups is contacted with endothelial cells which adhere to the substrate. The density of cells in the contacting medium is sufficient to cause the adhering cells forming a confluent layer on the substrate without cell proliferation. The substrate is polymeric.

0018Another aspect of the invention is an antithrombogenic article comprising a plasma-treated substrate having a confluent layer of adhered endothelial cells thereon according to claim 5. The articles are polymeric and may be of any shape. The preferred article is a conduit having an inside diameter of 0.5 mm or greater. One preferred, specific embodiment is a conduit having an inside diameter 2 to 6 mm. The lumen wall of these conduits is plasma-treated and endothelialized.

0019The plasma may be generated from any source of nitrogen which can be ionized. Preferred plasmas are generated from ammonia, most preferably ammonia containing a low concentration of oxygen.

0020The plasma treated substrate may be contacted with the endothelial cells in a suitable medium, such as a buffered saline, preferably under incubating conditions, to cause formation of an adhering confluent layer of the cells on the substrate.

0021Thus, in accordance with the invention articles having a plasma-treated polymeric surface and a confluent layer of adherent endothelial cells thereon are prepared by a two-step method which includes pretreatment of the article surface with a plasma to give an amine-rich surface and application of endothelial cells thereto. The plasma-treated surface prior to endothelialization is stable indefinitely and thus has a long shelf-life. Since the cells adhere quickly and become confluent without cell proliferation, a prosthetic device having the plasma-treated surface of the invention may be removed from stock, endothelialized, and be ready for implantation within minutes of procurement of sufficient cells. The advantages of speed to a patient undergoing surgery are apparent.

0022While this invention is satisfied by embodiments in many different forms, there will herein be described in detail preferred embodiments of the invention, with the understanding that the present disclosure is to be considered as exemplary of the principles of the invention and is not intended to limit the invention to the embodiments described.

0023One aspect of the present invention is a method for irreversibly modifying the surfaces of organic and inorganic substrates. More particularly, in accordance with the present invention, the surfaces of organic and inorganic substrates are irreversibly modified by bonding specific chemical functional species onto the surface of the substrate by contacting such surfaces with a plasma of a vaporized material in order to facilitate cell deposition and adherence.

0024The present invention can be employed to alter the surfaces of solid polymeric materials, including natural and synthetic addition and condensation polymers. Such polymeric materials include, but are not limited to, polyolefins, such as polyethylene, polypropylene, polyisobutylene and ethylene-alphaolefin copolymers, acrylic polymers and copolymers, such as polyacrylate, polymethylmethacrylate, polyethylacrylate; vinyl halide polymers and copolymers, such as polyvinyl chloride; polyvinyl ethers, such as polyvinyl methyl ether; polyvinylidene halides, such as polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile, polyvinyl ketones; polyvinyl aromatics, such as polystyrene, polyvinyl esters, such as polyvinyl acetate; copolymers of vinyl monomers with each other and olefins, such as ethylene-methyl methacrylate copolymers, acrylonitrile-styrene copolymers, ABS resins, and ethylene-vinyl acetate copolymers; natural and synthetic rubbers, including butadiene-styrene copolymers, poly-isoprene, synthetic polyisoprene, polybutadiene, butadiene-acrylonitrile copolymers, polychloroprene rubbers, polyisobutylene rubber, ethylene-propylenediene rubbers, isobutylene-isoprene copolymers and polyurethane rubbers; polyamides, such as Nylon 66 and polycaprolactam; polyesters such as polyethylene terephthalate, alkyd resins; phenol-formaldehyde resins; urea-formaldehyde resins, melamine-formaldehyde resins, polycarbonates; polyoxymethylenes; polyimides; polyethers; epoxy resins, polyurethanes; wool; cotton; silk; rayon; rayon-triacetate; cellulose, cellulose acetate, cellulose butyrate; cellulose acetate-butyrate; cellophane; cellulose nitrate; cellulose propionate; cellulose ethers; and carboxymethyl cellulose.

0025The substrates may be any shape, relatively flat or curved, and may be of any composition, such as continuous or particulate, porous or impervious, and large or small. The invention can be employed for altering the surfaces of crystals, powders, plates, strips, films, sheets, wire, fibers, fabrics, filaments, tubing, and cast, extruded or compressed articles, and the like.

0026For most plasma treatments in accordance with the present invention, a conventional plasma generator may be used. Such generators may include thermal, radio frequency, direct current, audio frequency, and microwave plasmas using internal and external capacitive coupling, inductive coupling, resistive coupling, and waveguide techniques. Electrical excitation may be provided by means of a DC or low frequency AC glow discharge produced by internal electrodes or coupling using inductive or capacitive means with higher frequency power sources from audio frequencies up through radio frequency and into microwave frequencies. Microwave waveguide techniques may also be used. Exemplary of suitable plasma generators are the plasma reactors made by Branson/IPC and plasma surface treatment systems made by Plasma Science Inc.

0027For the preferred embodiment of the invention wherein a small diameter conduit is treated with plasma, an apparatus as disclosed in EP-A-0 348 690 may be used.

0028The plasma may be generated from any gas or gaseous mixture which provides reactive functional groups from the gas grafted onto the surface. Suitable gases are, for example, oxygen, nitrogen and low molecular weight organic or inorganic compounds of oxygen or nitrogen such as methanol, acetonitrile or hydrogen cyanide. Preferred gases are nitrogenous such as nitrogen, aliphatic amines of from 1 to 6 carbon atoms, hydrazine, ammonia or mixtures thereof. Other components may be present in the gas mixture such as are hydrogen, halogen and inert gases such as helium, oxygen, neon, krypton and xenon.

0029The preferred plasma for surface modification in preparation for cell adherence is generated from ammonia containing a low concentration of oxygen. The oxygen to ammonia ratio may be from about 0.005 to 0.8, preferably from about 0.01 to 0.1. The desired ratio may be achieved by bleeding appropriate quantities of the gases into the reaction chamber after conventional pumpdown. However, for most surface treatments in accordance with the present invention, sufficient oxygen remains in the vacuum chamber after pumpdown to achieve the desired ratio. It is evident that the quantity of oxygen remaining in the chamber is related to the pumpdown pressure in the evacuated chamber. Thus a desired oxygen-nitrogen ratio within an appropriate total gas pressure may be obtained by evacuating the chamber to a pre-determined pressure and bleeding in the required amount of ammonia. In general, a close approximation of the desired oxygen concentration can be achieved merely by control of the pumpdown time.

0030Plasma generation for surface modification in accordance with the present invention may be carried out with a wide range of power settings, radio frequencies, durations of exposure, temperatures, and gas pressures. Ranges for these parameters which provide advantageous results are measured DC or AC power density levels of from 0.001 to 400 watts per cubic centimeter, oscillation frequencies up to 100 megahertz, 2 seconds to 12 hours, 0 to 200° C, and 1.3 x 10⁻⁵ to 1.3 x 10⁻¹ bar (0.01 to 100 torr). Preferred ranges for these parameters are .01 to 200 watts per cubic centimeter, 5 to 30 megahertz, 5 seconds to 120 minutes, 10-50° C, and 1.3 x 10⁻⁵ to 2.6 x 10⁻² bar (0.01 to 20 torr). Gas flow rates may vary from stagnant conditions to several volume replacements per second.

0031The pumpdown pressure which controls the oxygen concentration may be from about 0.1 to 100, preferably, about 6.6 x 10⁻⁶ to 6.6 x 10⁻⁵ bar (5 to 50 millitorr). Depending on the capacity of the pump, these pumpdown pressures may be reached in about 1 min. to 72 hours.

0032The polymeric surface of the invention modified by exposure to a plasma generated from an ammonia-oxygen mixture contains amino groups bonded onto its surface. In addition, oxygen containing groups, such as carbonyl, carboxyl, hydroxyl, ether and peroxide groups are present consequent to reaction of the surface with reactive oxygen species.

0033In the following examples, polymeric substrates are subjected to a plasma generated from an oxygen-ammonia mixture. The plasma-treated substrate surfaces may be examined by conventional electron spectroscopy for chemical analysis (ESCA), to measure binding energy shifts and determine therefrom the nature of the functional groups on the surface. Comparative ESCA data for the plasma-modified surfaces of the invention and untreated surfaces are given in Table I.

0034The amino and oxygen-containing groups introduced onto the substrate surface by the plasma render the surface amenable to the deposition and adherence of cells. In the second step of the method of the invention, endothelial cell types may be applied to the plasma treated surfaces of the invention. Methods to isolate and purify endothelial cells from various sources are well known, and the provision of cells for adherence to the plasma-treated substrate surface of the invention is not part of the invention.

0035Endothelialization may be carried out by contacting the plasma-treated substrate surface with sufficient cells suspended in a medium to cause formation of an adhered confluent layer without a requirement for cell proliferation. In accordance with the invention, this technique and the suspending medium are referred to as sodding and the sodding medium respectively.

0036Any sodding medium which is not deleterious to the cells may be used. Preferred sodding media are buffers, as for example, buffered saline. The cells adhere confluently in a manner of minutes, generally in about 1 to 60 minutes. If desired, adherence may be hastened by incubating the sodded surface at a temperature of from about 10 to 50° C, preferably at 37° C.

0037The number of cells required to sod the plasma-treated surface varies somewhat in accordance with the particular polymer and the particular surface chemistry after plasma treatment. In general about 10⁵ cells per square centimeter of surface to be covered is sufficient, but a greater concentration may also be used. Determination of this number and the concentration of cells in the sodding medium, which is not critical, are well within the purview of one skilled in the art. Likewise, determination of cell adherence and confluency by scanning electron microscopy or light microscopy after staining are conventional.

0038In another aspect of the invention, antithrombogenic articles having a plasma-treated surface and a confluent layer of endothelial cells adhered directly to the plasma-treated surface are provided. Exemplary of articles contemplated to fall within the scope of the invention are prosthetic devices which come into contact with blood, and include, as non-limiting examples, artificial hearts, heart valves, pins and preferably, vascular grafts. The most preferred article of the invention is a small diameter vascular graft, such as a conduit of inside diameter of 6 mm or less having a plasma-treated and endothelialized lumen wall.

Example I

0039Flat polyurethane film samples were modified in an RF plasma discharge system. A pair of 20.3 cm (8 inch) diameter aluminum electrodes spaced 9.5 cm (3 3/4 inches) apart were used inside a 30.5 cm (12 inch) diameter 55.8 cm (22 inch) tall vacuum chamber. RF power was delivered from a variable frequency oscillator/amplifier pair into a "T" matching network, through a balun transformer into the vacuum chamber using a sealed feedthrough, and then the balanced lines were connected to the opposing horizontal electrodes. The film samples were placed atop the lower electrode.

0040The chamber was pumped down to 60 mTorr over a period of 6 minutes. While continuing to pump, anhydrous ammonia gas was bled through a fine metering valve into the chamber at a rate sufficient to maintain a 2.6 x 10⁻¹ bar (200 mTorr) pressure. After thus purging the system for 1 minute, a 10.5 MHz 25 watt RF plasma was produced for 2 minutes to treat the film. Following treatment, the system was vented to atmosphere and the samples removed.

0041The surface chemistry of the samples was measured using ESCA and the resulting elemental compositions for treated and untreated samples are given in Table I. <tables id="tabl0001" num="0001"><table frame="all"><title>Table I</title><tgroup cols="7" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="22.50mm" /><colspec colnum="2" colname="col2" colwidth="22.50mm" /><colspec colnum="3" colname="col3" colwidth="22.50mm" /><colspec colnum="4" colname="col4" colwidth="22.50mm" /><colspec colnum="5" colname="col5" colwidth="22.50mm" /><colspec colnum="6" colname="col6" colwidth="22.50mm" /><colspec colnum="7" colname="col7" colwidth="22.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col7" align="center">Elemental Atomic Percent Values for Plasma Treated and Virgin Polyurethanes</entry></row><row><entry namest="col1" nameend="col1" align="left">Substrate</entry><entry namest="col2" nameend="col4" align="center">Plasma Treated</entry><entry namest="col5" nameend="col7" align="center">Virgin Polymer</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">Carbon</entry><entry namest="col3" nameend="col3" align="center">Oxygen</entry><entry namest="col4" nameend="col4" align="center">Nitrogen</entry><entry namest="col5" nameend="col5" align="center">Carbon</entry><entry namest="col6" nameend="col6" align="center">Oxygen</entry><entry namest="col7" nameend="col7" align="center">Nitrogen</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">Polyurethane I</entry><entry namest="col2" nameend="col2" align="right">71</entry><entry namest="col3" nameend="col3" align="right">18</entry><entry namest="col4" nameend="col4" align="left">7</entry><entry namest="col5" nameend="col5" align="right">83</entry><entry namest="col6" nameend="col6" align="left">12</entry><entry namest="col7" nameend="col7" align="char" char=".">3.9</entry></row><row><entry namest="col1" nameend="col1" align="left">Polyurethane II</entry><entry namest="col2" nameend="col2" align="right">77</entry><entry namest="col3" nameend="col3" align="right">12</entry><entry namest="col4" nameend="col4" align="left">9.3</entry><entry namest="col5" nameend="col5" align="right">91</entry><entry namest="col6" nameend="col6" align="left">4.5</entry><entry namest="col7" nameend="col7" align="char" char=".">4.2</entry></row><row><entry namest="col1" nameend="col1" align="left">Polyurethane III</entry><entry namest="col2" nameend="col2" align="right">72</entry><entry namest="col3" nameend="col3" align="right">18</entry><entry namest="col4" nameend="col4" align="left">7.7</entry><entry namest="col5" nameend="col5" align="right">74</entry><entry namest="col6" nameend="col6" align="left">20</entry><entry namest="col7" nameend="col7" align="char" char=".">5.1</entry></row><row><entry namest="col1" nameend="col1" align="left">Polyurethane IV</entry><entry namest="col2" nameend="col2" align="right">67</entry><entry namest="col3" nameend="col3" align="right">19</entry><entry namest="col4" nameend="col4" align="left">11</entry><entry namest="col5" nameend="col5" align="right">76</entry><entry namest="col6" nameend="col6" align="left">20</entry><entry namest="col7" nameend="col7" align="char" char=".">3.9</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">Polyurethane V</entry><entry namest="col2" nameend="col2" align="right">72</entry><entry namest="col3" nameend="col3" align="right">14</entry><entry namest="col4" nameend="col4" align="left">7.9</entry><entry namest="col5" nameend="col5" align="right">86</entry><entry namest="col6" nameend="col6" align="left">8.2</entry><entry namest="col7" nameend="col7" align="char" char=".">3.6</entry></row></tbody></tgroup></table></tables>

Example II

Plasma Treatment of a Small Diameter Polystyrene Conduit

0042The inside wall of a section of polystyrene tubing 15 cm long and a 2.5 mm inside diameter was subjected to a plasma generated in the plasma generator of EP-A-0 348 690. Conditions of plasma treatment were similar to those used in Example I except that a 11.4 MHz 75 watt RF plasma was used and ammonia gas pressure was maintained at 1.86 x 10⁻² bar (14 Torr). Treatment duration was 25 seconds.

Example III

Plasma Treatment of a Small Diameter Polyurethane Conduit

0043The inside wall of a section of polyurethane conduit 100 cm long and 3.5 mm inside diameter was plasma modified as described in Example II.

Example IV

General Procedure for Deposition of Endothelial Cells onto a Polymer Surface

0044A plasma treated polymer film was cut into disks having an area of 2 cm², and the disks were placed into the wells of a FALCON® 24-well tissue culture plate. A series of experimental controls was also prepared by adding one milliliter of 1% gelatin to each of six wells and one ml of 0.9% saline to six other wells. One ml of 0.9% saline was added to each well containing a plasma treated polymer disk. Both 24-well plates were covered with parafilm and maintained at 4°C for 24 hours to ensure adequate hydration of test and control surfaces.

0045The gelatin and saline solutions were removed by aspiration and 0.8 ml of a stock suspension containing 2.5 x 10⁵ endothelial cells per ml was added to the wells. The cells were incubated for one hour at 37°C in a 5% carbon dioxide atmosphere, then washed with buffer and decanted using trypsin. The test surfaces and controls were washed again and the adherent cells were stained with hematoxylin. The number of adherent cells was determined using standard light microscopy techniques.

0046The results of this experiment are given in Table II as adherent endothelial cell population density, in cells per cm² for a variety of plasma treated polymer formulations. <tables id="tabl0002" num="0002"><img file="EP0348969B1_D0001.tif" /></tables><tables id="tabl0003" num="0003"><img file="EP0348969B1_D0002.tif" /></tables>

Example V

Effect of Plasma Treatment of the Adherence of Primary Endothelial Cells

0047Following the general procedure for deposition of endothelial cells onto a polymer surface described in Example IV, an inoculum of primary human endothelial cells was applied to plasma treated and virgin polyurethane disk samples. Two polyurethane types were examined, with cell adherence results shown in Table III. While primary endothelial cells do not exhibit the same propensity for attachment to polymer surfaces as cultured endothelial cells, the relative affinity for plasma treated polymer surfaces is higher than for untreated virgin polymer surfaces. <tables id="tabl0004" num="0004"><table frame="all"><title>Table III</title><tgroup cols="2" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="78.75mm" /><colspec colnum="2" colname="col2" colwidth="78.75mm" /><thead valign="top"><row><entry namest="col1" nameend="col2" align="center">Primary Endothelial Cell Adherence to Plasma Treated and Virgin Polyurethane</entry></row><row><entry namest="col1" nameend="col1" align="left">Polymer</entry><entry namest="col2" nameend="col2" align="center">Adherent Cell Density (cells/cm²) mean ± standard deviation</entry></row><row><entry namest="col1" nameend="col1" align="left">Polyurethane A</entry><entry namest="col2" nameend="col2" /></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">plasma treated</entry><entry namest="col2" nameend="col2" align="left">14606 ± 7211</entry></row><row><entry namest="col1" nameend="col1" align="left">untreated virgin</entry><entry namest="col2" nameend="col2" align="left">6287 ± 2183</entry></row></tbody></tgroup><tgroup cols="2" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="78.75mm" /><colspec colnum="2" colname="col2" colwidth="78.75mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="left">Polyurethane B</entry><entry namest="col2" nameend="col2" /></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">plasma treated</entry><entry namest="col2" nameend="col2" align="left">15058 ± 7607</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">untreated virgin</entry><entry namest="col2" nameend="col2" align="left">7005 ± 5301</entry></row></tbody></tgroup></table></tables>

Example VI

Deposition of Cells Onto the Inside Wall of a Plasma

Treated Small Diameter Polyurethane Conduit

0048Polyurethane conduits of Example III with internal diameter of 3.5 mm were plasma treated using the conditions described in Examples I and II. A primary endothelial cell population was suspended in buffer as described in Example IV at an inoculation density of 2 x 10⁵ endothelial cells per square centimeter of polymer surface area calculated for the internal lumen of the conduits.

0049Each tube was encased in a supporting glass tube with connecting ports into which the cell suspension and buffer were introduced. The tubes were divided into two groups for incubation. Group I was rotated axially at a constant rate of 360 per minute and Group II was rotated axially in increments of 90 every fifteen minutes. Both groups were incubated for one hour at 37°C in a 5% carbon dioxide atmosphere. A gelatin control was also prepared according to the procedure of Example IV.

0050The cell adherence results appear in Table IV. Both methods of incubation allow endothelial cell adherence, however, a more uniform layer is achieved using the constant rotational method. <tables id="tabl0005" num="0005"><table frame="all"><title>Table IV</title><tgroup cols="3" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col3" align="center">Endothelial Cell Adherence to Plasma Treated Polyurethane Conduits</entry></row><row><entry namest="col1" nameend="col1" align="center">Conduit Sample Number</entry><entry namest="col2" nameend="col3" align="center">Adherent Cell Density (cells/cm²)</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">Group I (constant rotation)</entry><entry namest="col3" nameend="col3" align="center">Group II (incremental rotation)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">Sample 1</entry><entry namest="col2" nameend="col2" align="right">20753</entry><entry namest="col3" nameend="col3" align="left">22429</entry></row><row><entry namest="col1" nameend="col1" align="left">Sample 2</entry><entry namest="col2" nameend="col2" align="right">34539</entry><entry namest="col3" nameend="col3" align="left">8211</entry></row><row><entry namest="col1" nameend="col1" align="left">Sample 3</entry><entry namest="col2" nameend="col2" align="right">14595</entry><entry namest="col3" nameend="col3" align="left">19397</entry></row><row><entry namest="col1" nameend="col1" align="left">Sample 4</entry><entry namest="col2" nameend="col2" align="right">21017</entry><entry namest="col3" nameend="col3" align="left">10414</entry></row><row rowsep="1"><entry namest="col1" nameend="col3" align="justify">Gelatin Control = 14652</entry></row></tbody></tgroup></table></tables>