Effect of plasma glow, glutaraldehyde and carbodiimide treatments on the enzymic degradation of poly (L-lactic acid) and poly (gamma-benzyl-L-glutamate) films.

The hydrolytic and enzymic degradation of poly(L-lactic acid) (PLA) and poly(gamma-benzyl L-glutamate) (PBGA) films, together with a series of surface treatments, were studied, as a function of exposure time. The degradation of these polymers was monitored by weight loss, contact angle, pH changes and tensile strength studies. Glutaraldehyde treatment retained the maximum strength of PLA in buffer, followed by carbodiimide, compared with control films. On the other hand, plasma glow reversed the effect. The ability of alpha-chymotrypsin, carboxypeptidase, ficin, esterase, bromelain and leucine aminopeptidase to modulate the degradation of PLA and PBGA was also investigated. Addition of these enzymes to the polymer-buffer system reduced the tensile strength of these polymers variably. Among the six enzymes studied, leucine aminopeptidase showed the highest enzymic effect on the degradation of the glutaraldehyde-treated and bare PLA or bare PBGA films. However, glutaraldehyde-cross-linked PLA demonstrated maximum stability in buffers or in all other enzyme systems studied compared with bare PLA. It is conceivable that surface treatments on these polymers might have altered their physical and chemical configuration and the subsequent degradation properties. Surface modifications may provide new ways of controlling the biodegradation of polymers for a variety of biomedical applications.

Effect of liposome-albumin coatings on ferric ion retention and release from chitosan beads.

Ferric chloride was embedded in a chitosan matrix to develop a prolonged-release form. The in vitro release profiles of ferric ions from chitosan beads were monitored in 0.1 M Tris-HCl buffer, pH 7.4, using a UV spectrophotometer. The amount of drug release was much higher initially, followed by a constant slow release profile for a prolonged period. The initial burst release was substantially modified with liposome and albumin coatings. From scanning electron microscope studies, it appears that the ferric ions diffuse out slowly to the dissolution medium through the micropores of the chitosan matrix. Further, the liposome forms a phospholipid membrane layer in the pores of chitosan beads and encapsulates the ferric ions within their vesicles and controls the release profile. The chitosan beads loaded with ferric ions substantially inhibited the polyurethane-associated calcification, in an in vitro model system. The released ferric ions, appeared to alter the protein-surface binding and improved the biocompatibility of the matrix. The results propose the possibility of modifying the polymer matrix to obtain a desired controlled release of the drug for a prolonged period.

Changes in protein adsorption on polycarbonate due to L-ascorbic acid.

When a synthetic material is introduced into blood, plasma proteins are rapidly adsorbed on to its surface, followed by the attachment of formed elements of blood, leading to thrombus formation. Previous research reported that vitamin C (L-ascorbic acid) prolongs the clotting time due to the formation of vitamin C-calcium complexes, which reduce the availability of Ca2+ ions for the clotting mechanism in in vitro conditions. Contact angle and platelet adhesion studies have indicated that vitamin C modifies the surface-protein interaction and surface-platelet binding at the interface. In this paper an increased thickness of protein layer deposited on the polycarbonate substrate has been observed in the presence of vitamin C (approximately 50 A) using ellipsometric measurements. Further polyacrylamide gel electrophoresis (PAG) and infrared attenuated total reflection spectroscopy (IR-ATR) techniques have provided a better understanding of the interfacial phenomena. It seems, that the adsorption of albumin is increased, relatively, in the presence of vitamin C, when compared with that of fibrinogen and gamma-globulin from an equal amount of protein mixture.

Influence of steroid hormones on protein-platelet interaction at the blood-polymer interface.

To develop artificial materials for prolonged use in the vascular system, the complicated process of surface-induced thrombosis needs to be better understood. Steroidal hormones have a profound role in thrombosis and haemostasis, although adequate studies are not available to demonstrate their part in the thromboembolic phenomena that occur at the blood-foreign material interface. We studied the interfacial phenomena of five steroid hormonal drugs, Sustanon, Menstrogen, Mixogen, Durabolin and Ovral and their interaction with proteins and platelets toward an artificial surface using contact angle, polyacrylamide gel electrophoresis, trace labelling methods, etc. This study demonstrates the effect of these hormones to modulate platelet-surface attachment in the presence of platelet inducers. The addition of steroid hormones to the polymer-protein system can inhibit the level of surface-bound albumin where the fibrinogen binding to an artificial surface has been enhanced or unaltered. Steroids also increase platelet-surface attachment to variable degrees. Prolonged use of steroids or the oestrogen-containing oral contraceptive agents may not be advisable for patients having an artificial implant in contact with blood.

Chitosan beads and granules for oral sustained delivery of nifedipine: in vitro studies.

Nifedipine was embedded in a chitosan matrix to develop a prolonged-release form. The in vitro release profiles of nifedipine from chitosan beads and microgranules were monitored by UV spectrophotometer. The studies were performed in a rotating shaker (100 rev min-1) in 0.1 M HCl buffer (pH 2.0) or 0.1 M phosphate buffer (pH 7.4). Comparison was made between drug-loaded microbeads and microgranules. The amount and percentage of drug release were much higher in HCl than in phosphate buffer, probably due to the salt formation of the matrix (chitosan hydrochloride) at acid pH. The release rate of nifedipine from chitosan matrix was slower for beads than granules. These findings suggest the possibility of modifying the formulations to obtain the desired controlled release of the drug in an oral sustained-delivery system.

Chitosan matrix for oral sustained delivery of ampicillin.

Ampicillin was embedded in a chitosan matrix to develop an oral release dosage form. The in vitro release profile of ampicillin from chitosan beads and microgranules of chitosan was monitored, as a function of time, using a UV Spectrophotometer. The releasing studies were performed in a rotating shaker at 100 r.p.m., containing 0.1 M HCI buffer, pH 2.0, or 0.1 M phosphate buffer, pH 7.4, solutions, and a comparison was made between the drug loaded microbeads and microgranules. It seems that the amount and percentage of drug release was much higher in HCI solution compared with the phosphate solution, probably due to the gelation properties of the matrix at acid pH. The release rate of ampicillin from the chitosan matrix was slower for the beads as compared with the granules. From scanning electron microscopic studies, it appears that the drug forms a crystal structure within the chitosan beads, which dissolves out slowly to the dissolution medium through the micropores of the chitosan matrix. The results propose the possibility of modifying the formulation in order to obtain the desired controlled release of the drug for a convenient oral sustained delivery system.

Development of chitosan/polyethylene vinyl acetate co-matrix: controlled release of aspirin-heparin for preventing cardiovascular thrombosis.

Aspirin and heparin were embedded in chitosan/polyethylene vinyl acetate co-matrix to develop a prolonged release form. The in vitro release profiles of these drugs from the co-matrix system were monitored in Tris HCl buffer pH 7.4, using a UV spectrophotometer. The amount of drug release was initially much higher. followed by a constant slow release profile for a prolonged period. The initial burst release was substantially modified with styrenebutadiene coatings. From scanning electron microscopy studies it appears that the drugs diffuse out slowly to the dissolution medium through the micropores of the co-matrix. The released aspirin-heparin from the co-matrix system had shown their antiplatelet and anticoagulant functions. The results propose the possibility of delivering drug combinations, having synergestic effects for therapeutic applications.

Use of plasma glow for surface-engineering biomolecules to enhance bloodcompatibility of Dacron and PTFE vascular prosthesis.

The search for a nonthrombogenic material having patency to be used for small diameter vascular graft applications continues to be a field of extensive investigation. The purpose of the present study was to examine whether surface modification of polytetra fluoroethylene (PTFE, Teflon) and polyethylene-terephthalate (Dacron) vascular grafts might extend graft biocompatibility without modifying the graft structure. A series of surface coatings were prepared by modifying the argon plasma-treated PTFE and Dacron grafts with collagen IV and laminin and subsequently immobilizing bioactive molecules like PGE1, heparin or phosphatidyl choline via the carbodiimide functionalities. Surface analysis by Fourier transform infrared spectroscopy-attenuated total reflectance revealed the presence of new functional groups on the modified graft surfaces. In vitro studies showed that fibrinogen adsorption and platelet adhesion on modified grafts were significantly reduced. This study proposes that surface grafting of matrix components (collagen-type IV and laminin) and subsequent immobilization of bioactive molecules (PGE1, heparin or phosphatidyl choline) changed the surface conditioning of vascular grafts and subsequently improved their biocompatibility. However, more detailed in vivo studies are needed to confirm these observations.

Structural studies on bovine bioprosthetic tissues and their in vivo calcification: prevention via drug delivery.

Cardiovascular calcification, the formation of calcium phosphate deposits in cardiovascular tissue, is a common end-stage phenomenon affecting a wide variety of bioprostheses. To study the process of calcification in tissue prosthetics, glutaraldehyde-treated bovine pericardium, dura mater and fascialata were implanted subcutaneously in rats and retrieved 21 days later and thereby morphological findings were correlated with biochemically determined levels of calcium. Transmission electron microscopy showed that calcification primarily involved the surface of collagen fibrils and the interfibrillar spaces. The deposition of calcium was higher with dura and fascia prostheses compared to pericardium. However, the release of Fe3+ ions from chitosan matrix had substantially inhibited the deposits of calcium in all implanted tissues. It seems that the structural and anatomical features of the tissue is one of the important factors for tissue-associated calcification. It is also confirmed that glutaraldehyde-preserved pericardium is the most suitable material for the development of cardiac prosthesis, with an appropriate drug combination therapy for prevention of pathological calcification.

Protein/platelet interaction with an artificial surface: effect of vitamins and platelet inhibitors.

Protein adsorption and platelet adhesion are two important biological processes arising at the blood-prosthetic interface. The effect of Vitamins and antiplatelet drugs to modulate the surface induced platelet adhesion to polycarbonate was investigated using washed calf platelets in presence and absence of fibrinogen. This study also demonstrated the effects of Vitamins and antiplatelet drugs towards protein adsorption to an artificial surface. It seems Vitamin B6, Vitamin E, combinations of Aspirin-Persantine, Aspirin-Vitamin C, a synthetic Polyelectrolyte and Galactosamine reduced the fibrinogen (fg) surface concentration from a mixture of proteins. These antiplatelet agents also enhanced the albumin surface concentration. This itself may be one of the parameters to reduce the platelet adhesion towards an artificial surface. A combination of Aspirin-Vitamin C-Vitamin B6-Vitamin E inhibited the fibrinogen surface binding, which might be beneficial to improve the blood compatibility of an artificial surface.

Structural relevance of adsorbed aminosugars on polycarbonate - antithrombogenicity.

The effects of glucosamine, galactosamine, and mannosamine on platelet adhesion and coagulation induced by an artificial surface were investigated. Glucosamine and galactosamine showed both antiplatelet and anticoagulant effects, but in case of mannosamine none of the above effects was found significant. The three analogs studied were having the same molecular weight and common molecular structure, however from our preliminary observations it could be suggested, that the spatial arrangement of functional groups in the glucopyranose ring at C2, C4 positions were important for it's activity.

Activated charcoal microcapsules and their applications.

Activated charcoal, long known to the ancients as a substance of therapeutic value in a variety of maladies, has recently been "rediscovered" to be of great value in medical applications. Activated charcoal hemoperfusion is effective in blood purification for removal of various circulating toxic materials and waste metabolites, directly. However, particulate release and platelet adhesion prevent its continued clinical use. Polymeric coatings or microencapsulation of charcoal within polymers have improved their blood compatibility. Chitosan encapsulated activated charcoal (ACCB) beads have been extensively investigated in our group for the removal of various toxins such as urea, creatinine, uric acid, bilirubin, etc. This article highlights various methods of microencapsulation procedures of activated charcoal and the importance of this novel material for a variety of biomedical applications. Further, this review provides an insight to the future perspectives for using them in clinical practice.

Phenyl alanine, tryptophan immobilized chitosan beads as adsorbents for selective removal of immunoproteins.

The use of adsorbents for the treatment of patients suffering from various immune diseases is still in its infancy. Therefore, the development of selective absorbents for the removal or decrease of immunoproteins from plasma is of great importance. In this study, chitosan, a natural polysaccharide having structural characteristics similar to glycosamino glycans, which is non-toxic and biocompatible, has been used for protein adsorption studies. Amino acids like phenyl alanine and tryptophan in different ratios are bonded to these polymers to observe immunoadsorption. Several layers of phenyl alanine or tryptophan have been coated covalently on chitosan beads using N2-plasma, carbodiimide or glutaraldehyde treatments. Scanning electron micrographs have revealed the surface morphological changes after such modifications. The surface modified chitosan beads have exhibited high binding affinity for gamma-globulin compared to bare beads. It is also observed that the amount of fibrinogen adsorption is reduced on modified substrate. A selective removal of IgG and IgM has also been observed with these modified matrix when tested with human plasma, using immuno diffusion methods. The modified chitosan membranes have demonstrated a reduction in platelet attachment, showing that these substrates have become more blood compatible. Hence, it appears that modified chitosan surfaces may be an excellent sorbent system for haemoperfusion due to their high binding affinity for immunoproteins and blood compatibility. Further studies are needed to determine the behaviour under clinical conditions.

Influence of polyethylene glycol graftings on the in vitro degradation and calcification of bovine pericardium.

Calcification is a frequent cause of the clinical failure of bio-prosthetic heart valves fabricated from glutaraldehyde pretreated bovine pericardium (GATBP). This article reports on various chemical techniques for grafting polyethylene glycol (PEG) on bovine pericardium, their biostability, and calcification. The process of calcification profile was studied by in vitro experiments via the incubation of pericardial samples in a metastable solution of calcium phosphate. The calcification profile of PEG-modified bovine pericardium through glutaraldehyde linkages was significantly reduced compared to other methods of grafting. The mechanical property of these PEG-modified tissues after enzyme (collagenase) digestion and calcification were also investigated. PEG grafting of BP via glutaraldehyde or hexamethylene diisocyanate had shown better mechanical stability compared to other grafting methods used. In conclusion, it seems that the surface modification of bovine pericardium through high molecular weight PEGs via glutaraldehyde linkages may provide new ways of controlling tissue biodegradation and calcification.

Platelet adhesion and spreading on protein-coated surfaces: variations in behavior in washed cells, PRP, and whole blood.

Platelet attachment and spreading were monitored on glass and various protein coated glass, under shear with washed platelets, platelet rich plasma (PRP) and whole blood, using fluorescence Optimas imaging system and software. Results showed that the platelet adhesion and spreading were sensitive to the nature of precoated proteins and the type of medium used for introducing platelet suspension for the study. In general, the cell adhesion and spreading were higher with fibrinogen (Fg), fibronectin (Fn), von Willebrand Factor (vWF), and collagen precoated surfaces. In the presence of albumin on the surface, however, platelets could not attach and spread fully when using washed cells. But, the surface attachment and spreading of the cells were higher on albumin substrates on exposure to PRP or whole blood. This may be due to the replacement of precoated albumin by other plasma proteins, like Fg to facilitate the platelet-surface attachment. The composition of this layer determines the extent of platelet activation and the adhesive strength between platelets and polymer surface. These results indicate that multiple adhesion receptors can mediate platelet adhesion and spread to matrix proteins immobilized on surfaces. Further, these studies combined with some of our earlier observations and suggestions propose the need for developing in vitro tests that resemble in vivo conditions.

Influence of steroid hormones on bovine pericardial calcification.

Calcification is a frequent cause of the clinical failure of bioprosthetic heart valves fabricated from glutaraldehyde pretreated bovine pericardium (GABP). The present investigation describes the influence of steroid hormones in the mineralization of GABP, in an extra-circulatory environment. Calcification was studied on GABP incubation in a metastable solution of calcium phosphate containing steroid hormones such as estrone, progesterone, 7(OH) progesterone, testosterone and beta-estradiol. It is interesting to note that certain steroids can variably increase the GABP calcification. Further, the effect of these steroids in an in vitro hydroxyapatite (HA) formation was investigated. In addition, we observed these steroids alter the calcium transport through GABP in diffusion experiments and also in HA formation. Therefore, it is conceivable that prolonged use of steroids or steroids containing oral contraceptive agents may not be advisable for patients having bioprosthetic implants in contact with blood. A better understanding of the mechanism of these drugs under in vivo conditions is needed to develop applications.

The antithrombotic versus calcium antagonistic effects of polyethylene glycol grafted bovine pericardium.

Cardiovascular calcification, the formation of calcium phosphate deposits in cardiovascular tissue, is a common end stage phenomenon affecting a wide variety of bioprosthesis. This study proposes a novel approach of reducing pericardial calcification and thrombosis via coupling polyethylene glycols (PEG) to glutaraldehyde treated bovine pericardium via acetal linkages. The calcification of the PEG modified tissue and the control pericardium (extracted and glutaraldehyde treated) was investigated by in vivo rat subcutaneous implantation models and by in vitro meta stable calcium phosphate solutions. Scanning electron microscopy showed that calcification primarily involved the surface of collagen fibrils and the intrafibrillar spaces. However, the grafting of pericardium with PEG-20,000 had dramatically modified the surface and subsequently inhibited the deposits of calcium. Further, the modified tissue had also reduced the platelet surface attachment. Such a reduced calcification of PEG modified tissues can be explained by decrease of free aldehyde groups, a space filling effect and therefore improved biostability and synergistic blood compatible effects of PEG after coupling to the tissues. This simple method can be a useful anticalcification treatment for implantable tissue valves.

Inhibition of platelet adhesion to glow discharge modified surfaces.

Plasma glow technique has created much interest in the field of surface modification of polymers due to its versatility of generating active polar groups on the surface without affecting the bulk properties. Here an attempt is made to inter-relate the surface properties and platelet adhesion on various polymeric substrates due to plasma treatments. Initially, a critical review of the process and development of thrombosis upon contact of an artificial surface with blood, has been provided, which has been extended with the need for surface modifications to improve their blood compatibility and the versatility of plasma treatments for such modifications have been emphasized. Phospholipids like phosphoryl choline, phosphatidyl choline and phosphoryl ethanolamine were attached to Angioflex surface by plasma glow. The role of such modified substrates to interact with platelets were investigated using Tyrode washed calf platelets. It seems, glow discharge modified phosphoryl choline bilayers dramatically inhibited the platelet-surface binding, which may be due to their biochemical resemblance with thromboresistant surfaces of human blood cells. Further, the behaviour of all phospholipids towards bloodpolymer interaction is not similar and may change depending on the nature of their functional groups, net charge of the phospholipid adsorbed surface and their interaction with platelets and its activation. It is possible to chemically immobilize lipid bilayers on standard polymers, using plasma glow, to improve their biological performance; by suitably selecting the phospholipid combinations.

Inhibition of bioprosthesis calcification due to synergistic effect of Fe/Mg ions to polyethylene glycol grafted bovine pericardium.

Calcification has limited the durability of bioprosthetic heart valves fabricated from glutaraldehyde pretreated porcine aortic valves or bovine pericardium (BP). The present study describes calcium antagonistic effect of polyethylene glycol grafted bovine pericardium (PEG-GABP) with Fe2+/Mg2+ delivery from a co-matrix system in rat subcutaneous model. Retrieved samples were biochemically evaluated for calcification and alkaline phosphate (AP) activity. Scanning electron micrographs of 21-day explants had shown excessive calcification with glutaraldehyde treated BP (control). However, the PEG grafting and Fe/Mg release had substantially inhibited the deposition of calcium on BP. The extractable alkaline phosphatase activity was also reduced with PEG grafting and metal ion release to BP. The extractable AP had shown peak activity at 72 h [for GATBP--250.5 +/- 1.2 nm pnp/mg protein/min enzyme activity (unit), PEG-GABP--165.2 +/- 16.6 units], but markedly reduced after 21 days (22.1 +/- 1.8 and 12.0 +/- 1.5 units, respectively). The initial high levels may be due to tissue injury via surgery, which mitigated with time. It is assumed that ferric ions may slow down or retard the calcification process by the inhibition of proper formation of hydroxy apatite while magnesium ions disrupt the growth of these crystals by replacing Ca2+. In addition it maybe hypothesized that these metal ions may inhibit the key element alkaline phosphatase, which acts as the substrate for mineralization. Hence, it is conceivable that a combination therapy via surface grafting of PEG and local delivery of low levels of ferric and magnesium ions may prevent the bioprosthesis associated calcification.

Effects of double cross-linking technique on the enzymatic degradation and calcification of bovine pericardia.

The strength, resorption rates, and biocompatibility of collagenous biomaterials are profoundly influenced by the method of cross-linking. The in vitro and in vivo calcification and enzymatic degradation of bovine pericardia (BP) after a series of surface modifications were studied as a function of exposure time. Collagenase degradations of modified BP were monitored by scanning electron microscopy and tensile strength measurements. Bovine pericardium was modified by a combination of different tissue fixatives such as glutaraldehyde (GA), carbodiimide (EDC), diisocyanate (HMDIC), and polyethylene glycol (PEG). GA-PEG-EDC-PEG and GA-PEG-HMDIC-PEG combination treated BP retained maximum stability in collagenase digestion compared to GATBP. In vitro calcification studies and in vivo rat subcutaneous implantations of modified pericardium have shown substantial reduction in the calcification of double cross-linked BP with PEG modification. Further, the biocompatibility aspects of pericardial tissues were established by platelet adhesion and octane contact angle. It seems that cross-links involving amino and carboxyl residues may provide new ways of controlling biodegradation and calcification.