Rajender Sipehia

Biocompatibility aspects of new stent technology

Stent implantation represents a major step forward since the introduction of coronary angioplasty. As indications continue to expand, better understanding of the early and late biocompatibility issues appears critical. Persisting challenges to the use of intracoronary stents include the prevention of early thrombus formation and late neointima development. Different metals and designs have been evaluated in animal models and subsequently in patients. Polymer coatings have been proposed to improve the biocompatibility of metallic stents or to serve as matrix for drug delivery and they are currently undergoing clinical studies. The promises of a biodegradable stent have not yet been fulfilled although encouraging results have recently been reported. Continuous low dose-rate brachytherapy combining the scaffolding effect of the stent with localized radiation therapy has witnessed the development and early clinical testing of radioactive stents. The combined efforts of basic scientists and clinicians will undoubtedly contribute to the improvement of stent biocompatibility in the future.

Enhanced growth of animal and human endothelial cells on biodegradable polymers

Biodegradable polymers such as poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA) and PGA coated with PLLA are being employed for cell transplantation and for in vivo regeneration of vascular tissue. Ingrowth and organization of fibrovascular tissue inside polymer scaffolds lead to the occlusion of the regenerated blood vessel. In order to provide regulatory mechanisms to control the development of an inner capsule, endothelialization of these materials is necessary. To achieve this, we employed a novel ammonia plasma technique to surface modified PLLA substrates. Human endothelial cell (HUVEC) and rabbit microvascular endothelial cell (RbMVEC) growth was studied on modified PLLA and control PLLA. Our studies show that modified PLLA and fibronectin (Fn)-coated modified PLLA exhibited statistically significant improvement in HUVEC and RbMVEC growth (P<0.001) when compared to PLLA and Fn-coated PLLA. Therefore, ammonia plasma treatment gives us the unique capability of modifying prosthetic biomaterials of various constructs with the eventual transplantation of mammalian cells to be used in tissue engineering or as biological implants.

Antithrombotic and fibrinolytic system of human endothelial cells seeded on PTFE: the effects of surface modification of PTFE by ammonia plasma treatment and ECM protein coatings

The aim of this study was to determine the effects of ECM protein coatings and surface modification of PTFE on the ability of seeded human endothelial cells (EC) to secrete prostacyclin (PGI2), plasminogen inhibitor-1 (PAI-1) and tissue plasminogen activator (t-PA). PTFE surfaces were modified by a novel surface modification technique based on ammonia plasma. Fibronectin, collagen type-1 and gelatin-coated ammonia plasma modified PTFE and unmodified PTFE surfaces were employed and compared in this study. All ammonia plasma modified surfaces showed similar secretions of PGI2 compared to non-modified PTFE surfaces. With the exception of gelatin-coated modified PTFE, seeded EC seeded on all modified PTFE showed lower levels of PAI-1 secretion compared to those seeded on unmodified PTFE. The specific activity of t-PA secreted by EC seeded on ammonia plasma modified and fibronectin coated modified PTFE showed increases of 100 and 30%, respectively, when compared to their unmodified counterparts. Our studies show that EC seeded on modified PTFE have ability to secrete PGI2 that modulates the early phase of thrombus formation. Furthermore, superior t-PA profile, along with lower levels of PAl-1 suggest that ammonia plasma modification and use of appropriate ECM proteins can modulate antithrombotic and fibrinolytic properties of in vitro endothelialized vascular prostheses. Accordingly, these surfaces may be suitable to further develop protocols and other strategies for arterial and venous reconstruction.

ENHANCED ATTACHMENT AND GROWTH OF HUMAN ENDOTHELIAL CELLS DERIVED FROM UMBILICAL VEINS ON AMMONIA PLASMA MODIFIED SURFACES OF PTFE AND ePTFE SYNTHETIC VASCULAR GRAFT BIOMATERIALS.

Ammonia plasma generated by electrical discharge at low pressure was employed for the surface modification of PTFE and ePTFE. A new chemistry at the plasma treated surfaces is reported. X-ray photoelectron spectroscopy studies showed the incorporation of C-N, C-O, C = O etc functional groups on the plasma treated surfaces. Human endothelial cells derived from umbilical veins (HUEC) were used to seed the plasma treated PTFE and ePTFE surfaces to assess the attachment and growth. Enhanced attachment and growth of HUEC was observed on the plasma treated surfaces. In addition, the performance of these surfaces in this respect was found to be considerably superior to human collagen or human fibronectin or collagen-fibronectin coated PTFE. HUEC attachment and growth on these plasma treated surfaces was further enhanced by immobilizing collagen or fibronectin or collagen-fibronectin. Ammonia plasma treated and untreated ePTFE vascular graft samples were seeded with 3.6 X 10(4) cells/sample. At 24 hrs after seeding, HUEC cell attachment was studied. Although, HUEC attachment on collagen or fibronectin coated ePTFE was improved, but there was no significant difference between the number of cells attached to these surfaces when compared with those adhered to plasma treated ePTFE without collagen or fibronectin coating. Collagen or fibronectin coated plasma treated surfaces showed better performance over their respective controls.

Transplantation of Human Endothelial Cell Monolayer on Artificial Vascular Prosthesis: The Effect of Growth-Support Surface Chemistry, Cell Seeding Density, Ecm Protein Coating, and Growth Factors

The failure rates of synthetic vascular grafts, when placed in low blood flow environments in humans, are not acceptable. Thus, endothelial cell (EC) seeding technology of vascular grafts was developed to prepare prostheses lined with a human monolayer expressing optimal thromboresistant properties. In a clinical setting, endothelialization of a graft can be achieved using higher cell seeding densities, or by creating a surface on which EC can adhere and grow to confluence. But, human endothelial cells show little or no proliferation on the currently available graft materials. In this study, surface modification of PTFE and ePTFE by ammonia plasma treatment was carried out to enhance its interactions with ECM protein, EC growth factors, and with EC harvested from human umbilical vein (HUVEC), and from human saphenous veins (HSVEC). Our data shows that various vascular graft materials generated from ammonia plasma treated PTFE and ePTFE exhibited statistically significant improvements in HUVEC and HSVEC growth when compared to their respective controls (p values < 0.001). Growth of HSVEC on ammonia plasma treated ePTFE without ECM protein coating was also found to be statistically significant in comparison to that on fibronectin coated ePTFE (p < 0.001). The final HSVEC cell densities found on various ePTFE surfaces prepared from ammonia plasma treated ePTFE, suggests that transplantation of HSVEC monolayers on vascular prostheses can be established within clinically relevant times. Ammonia plasma treatment process provides an unique opportunity to surface modify prosthetic materials of various construct to transplant mammalian cells including those that have undergone ex vivo gene transfer, and to deliver angiogenic molecules to a target area for tissue development.

The enhanced attachment and growth of endothelial cells on anhydrous ammonia gaseous plasma modified surfaces of polystyrene and poly(tetrafluoroethylene)

Anhydrous ammonia gaseous plasma technique was used for the surface modification of polystyrene petri dishes and poly(tetrafluoroethylene) (PTFE) membranes. Amino groups were added onto surfaces by exposing them to ammonia plasma. Plasma modified polymeric surfaces and control polymeric surfaces were seeded with bovine pulmonary artery endothelial cells (EC). It was found that attachment of EC to control polystyrene surface was negligible. On the plasma modified polystyrene surface, there was improved attachment and growth of EC. At 96 hours, plasma modified surfaces yielded an order of 3 magnitudes more cells compared to those on control. Twenty four hours after seeding the cells, the percentage of EC attachment to control PTFE surfaces and modified surfaces were found to be about 36% and 92% respectively.

Albuminated Polymer Surfaces for Biomedical Application

Amino groups were added on to the surfaces of Celgard-2400 membranes by exposing them to an ammonia plasma. The presence of amino groups on the surfaces was detected by an attenuated total reflection Fourier Transform infrared spectrometer and by the Auger electron spectrometer. Through these amino groups, albumin was attached to the membranes. In some experiments, the attached albumin was further stabilized by cross-linking with glutaraldehyde. The effect of washing the albuminated membranes with saline and with plasma was investigated. It was observed that after the initial wash-out of albumin, the concentration of attached albumin tends to level off. The amount of albumin retained on the membranes varied between 275 to 357 micrograms/cm2.

X-ray Photoelectron Spectroscopy Studies, Surface Tension Measurements, Immobilization of Human Serum Albumin, Human Fibrinogen and Human Fibronectin onto Ammonia Plasma Treated Surfaces of Biomaterials Useful for Cardiovascular Implants and Artificial Cornea Implants

XPS studies of untreated and ammonia plasma treated surfaces of PTFE, ePTFE, Dacron, P(HEMA), PMMA, Silastic and PS were carried out. Ammonia plasma treatment caused significant changes in the surface composition. The curve-fitting results confirmed the incorporation of nitrogen and oxygen in the form of functional groups such as C-N, C=O, C-O, Si-N, Si-OH etc. Increases in the values of surface tension occurred. The surface tension of plasma treated surfaces varied between 44-48 erg/cm2 with the exception of Dacron which became wettable. Enhanced immobilization of human albumin on plasma treated surfaces was achieved. When washed with 0.2% Tween in buffer, these albuminated surfaces were found to be stable compared to control samples. Increased immobilization of human fibrinogen was also observed. The ammonia plasma treated surfaces showed high binding properties and retention for human fibronectin. Ionic interaction between proteins solution and plasma treated surfaces may be cause of the increase attachment of these biological molecules.

Ftir-Atr Spectra of Protein a Immobilized on to Functionalized Polypropylene Membranes by Gaseous Plasma of Oxygen and of Anhydrous Ammonia

Polypropylene membranes were treated in a gaseous plasma of oxygen or anhydrous ammonia, in order to add hydroxyl groups (oxygen plasma) or amino groups (ammonia plasma) on their surfaces. The presence of these functional groups was detected by FTIR-ATR spectrometry. The FTIR-ATR spectrum of free Protein A was recorded and absorption bands for amide I and amide II at 1650 cm-1 and 1552 cm-1 were identified. The surfaces of immobilized Protein A on to hydroxylated polypropylene membranes and on to polypropylene membranes having amino groups, were characterized by FTIR-ATR spectrometry. The presence of amide I and amide II absorption bands confirmed the immobilization of Protein A on to functionalized polypropylene membranes by gaseous plasma modification.

Enhanced oxidation of bilirubin by an immobilized tri-enzyme system of glucose oxidase, bilirubin oxidase and horseradish peroxidase

Bilirubin oxidase was immobilized to nylon fibres. A tri-enzyme system composed of glucose oxidase, bilirubin oxidase and horseradish peroxidase was also immobilized to the fibres. Both immobilized systems were tested and it was found that the latter gave enhanced oxidation rates for bilirubin.