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Modern metal processing for improved load-bearing surgical implants.

A review of modern methods for preparing metallic alloys that could be useful for the fabrication of load-bearing metallic biomaterials is presented. The use of rapid solidification processing and surface modification of metals by ion implantation or surface coatings and variations thereof is used currently for the formation of novel metallic alloys in other high-tech fields, notably the optoelectronics industry. Further studies to explore potential benefits for surgical implant fabrication through the application of these technologies is recommended.

Alloys

Liposome encapsulated hemoglobin: long-term storage stability and in vivo characterization.

Liposome Encapsulated Hemoglobin (LEH) has been the focus of research and development at the Naval Research Laboratory in an effort to find a viable oxygen-carrying resuscitative fluid. Previous reports from our laboratory have shown that LEH binds and releases oxygen in a manner similar to red blood cells, and that it can sustain life when red cell hematocrits are decreased to critical levels. We have also reported on LEH with regards to preparative methods, scale-up feasibility, toxicity, hemodynamics, hemoglobin P50 modification by coencapsulation of organic phosphates, liposomal surface modification, and storage strategies. In this report, the issue of LEH efficacy following long-term storage in the dry state will be addressed. We have shown that hemoglobin, liposomes, and LEH may be successfully lyophilized and rehydrated to viable states. The modification of the LEH formulation by addition of the carbohydrate trehalose results in the successful lyophilization and storage of LEH. In vitro characterization of LEH stored in the dry state for up to six months includes measurement of oxygen-carrying capacity, liposome size retention, methemoglobin production, and the intraliposomal hemoglobin concentration. The in vivo studies report on physiological parameters such as circulation persistence, blood chemistry, and pathological examination in mice.

Animals

Immobilization of enzymes on polypropylene bead surfaces by anhydrous ammonia gaseous plasma technique.

Anhydrous ammonia gaseous plasma technique was used for the surface modification of polypropylene beads. Amino groups were added onto the surfaces of beads by exposing them to ammonia plasma. Through these amino groups covalent immobilization of glucose oxidase and peroxidase were carried out. The total amounts of immobilized glucose oxidase and immobilized peroxidase were found to be 52 and 43 micrograms/cm2, respectively. To assess the stability of enzyme-polypropylene linkage, beads with covalently immobilized glucose oxidase and peroxidase were washed with phosphate buffer. It was found that after the removal of the adsorbed enzymes, the concentration of covalently immobilized enzymes tended to reach a steady state. After additional washing with buffer for 5 to 6 h, 40-55% of the immobilized enzymes were found to be in the active form.

Ammonia

Enhanced albumin binding to polypropylene beads via anhydrous ammonia gaseous plasma.

Plasma surface modification technique was used to add amino groups onto the surfaces of polypropylene beads by exposing them to anhydrous ammonia plasma. Through these amino groups, albumin was attached to the polypropylene beads. Attached albumin was further stabilized by crosslinking with glutaraldehyde. The effect of washing albuminated polypropylene beads with saline and human plasma was investigated. It was found that after initial rapid removal of albumin, the concentration of attached albumin tended to reach a steady-state. After 52 h of washing, the amount of albumin retained on the beads varied between 125 and 171 micrograms/cm2.

Albumins

Stainless steel mesh supports high density cell growth and production of recombinant müllerian inhibiting substances.

Stainless steel mesh supported the high density growth of anchorage dependent CHO fibroblasts without the use of a special culture system. CHO cells, designated B-9, containing an amplified genomic construct of the human gene for Müllerian Inhibiting Substance (MIS), grew to a high confluent density on stainless steel meshwork while producing substantial amounts of human recombinant MIS over a long period of time. The mesh could be easily coated with various extracellular matrix proteins, such as Laminin, Fibronectin, Collagen or Matrigel, which permitted the testing of the effects of surface modifications on cell yield and recombinant protein production. Since the amount of medium per surface area required for optimal cell growth is lower than for some large volume cell culture methods, media costs can be reduced using mesh. In addition, no special cell culture equipment or complex manipulations are required. Thus, the use of meshwork for anchorage-dependent cells can increase the efficiency of growth and decrease the cost of recombinant protein production.

Animals

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.

Ammonia

The biomechanical and histopathologic effects of surface texturing with silicone and polyurethane in tissue implantation and expansion.

There has been considerable interest in determining the effect of morphologic alterations of prosthetic surfaces on capsule response in breast surgery. The purpose of this study was to provide a precise, three-dimensional evaluation of soft-tissue response to surface modifications in both implantation and expansion. Expandable 100-cc prostheses were designed with one of three surfaces: textured silicone (Biocell), standard smooth silicone, or polyurethane (Natural-Y, Meme). A new submuscular implantation site in the rabbit was developed. Each animal randomly received a smooth-surface device on one side and either a textured silicone or polyurethane device on the other. In one group of animals, the prostheses were expanded monthly. Capsular response was evaluated monthly in vivo using standardized techniques as well as biomechanical methods for up to 6 months in the expander group (n = 7 to 16) and 8 months in the implant group (n = 7 to 15). Analysis of biomechanical and histologic data revealed that prosthetic surface morphology can specifically alter capsular response. Polyurethane was the only effective surface in preventing capsular contracture in implantation. In expansion, both textured silicone and polyurethane surfaces resulted in significantly less capsular contracture and less resistance to expansion than comparable smooth-surfaced controls. Statistical comparisons reveal that the biomechanical methods utilized in this study provide the most precise and objective method of defining overall soft-tissue contracture around implanted biomaterials.

Animals

Correlated scanning and transmission electron microscopy of developing lens vesicle in the chick.

Cells within the lens placode undergo transient surface modifications during the course of invagination. At stage 13, cellular apices bulge outward and display decreased surface diameters when compared to adjacent surface ectoderm. The lens vesicle is well invaginated by stage 14. An area of modified cells can be recognized ventral to the lens vesicle aperture. These cells present a smaller surface area and increased pleomorphism. Normal surface ectoderm extends to the dorsal and lateral edges of the aperture. The apical surfaces of cells which line the lumen of the lens vesicle display a complex topography such that individual cell boundaries are not easily distinguished. At stage 17, cellular processes which partially occlude the closing aperture of the lens vesicle may assist in reestablishing ectoderm over the newly formed lens. Preliminary results of cytochalasin B exposure on the invaginating lens vesicle suggest that the contractile action of microfilaments influences the complex cellular topography.

Animals

Polymers for biodegradable medical devices. VII. Hydroxybutyrate-hydroxyvalerate copolymers: degradation of copolymers and their blends with polysaccharides under in vitro physiological conditions.

The hydrolytic degradation of hydroxybutyrate-hydroxyvalerate copolymers was monitored in vitro at 37 degrees C and pH 7.4. Direct use of bulk properties such as weight loss and tensile strength did not reveal substantial changes in the polymer matrix over degradation periods of several months. Despite this, the polymers were demonstrated to undergo significant modification during this period, in ways that markedly influence their subsequent behaviour. Combined use of goniophotometry and surface energy measurements revealed that surface modification begins at an early stage and is accompanied by diffusion of water into the matrix and a progressive increase in polymer porosity. Relatively little change in the molecular weight and some increase in the crystallinity of the matrix occurred during these early months. As a result, the tensile strength of the polymer varies little in this period. As the porosity of the matrix increases, hydrolytic chain scission within the matrix and diffusion out of degradation products proceeds more effectively. Decrease in matrix molecular weight, increase in matrix erosion, weight loss and loss of tensile strength began at a much more dramatic rate. The apparent resistance of the polymer to degradation in the early months is followed by an accelerated degradation phase around and beyond 1 yr. The use of filters that can dissolve or hydrolytically degrade more rapidly than the hydroxybutyrate matrix accelerates the development of porosity within the matrix and thus enhances the decomposition process.

Biocompatible Materials

Corneal endothelial protection by heparin and sodium hyaluronate surface coating of PMMA intraocular lenses.

Protective effect on corneal endothelium from surface modification of PMMA intraocular lenses were evaluated. Sodium hyaluronate, heparin or both were covalently bound to the PMMA surface rendering it hydrophilic. Endothelial damage was evaluated by placing the intraocular lens optics on the endothelial surface of excised rabbit corneas. The type and extent of endothelial cell damage was evaluated by light microscopy after alizarine red/trypan blue staining and by scanning electron microscopy. The number of endothelial cells attached to the intraocular lens surface was counted in the light microscope after staining with haematoxylin--eosine. Significantly less damage (p less than 0.05) to the endothelium was caused by the surface modified hydrophilic intraocular lenses. There was no difference between the various types of surface coatings studied in this investigation.

Animals

ESCA investigation of low-temperature ammonia plasma-treated polyethylene substrate for immobilization of protein.

A low-temperature radiofrequency plasma excited in anhydrous ammonia was used to modify polyethylene substrate surfaces for covalent immobilization of proteins. Electron spectroscopy for chemical application (ESCA) was used for surface characterization of polyethylene to a depth scale of 7 nm. The data revealed that surface modification is extensive and occurs in seconds at low discharge power. Primary amino functionalities were detected on the polyethylene surface and the level is dependent on plasma parameters. 125I-labelled antibodies covalently attached to amino groups via glutaraldehyde allowed the conditions for optimum level of primary amine to be established. Both ESCA data and protein loadings are in excellent agreement.

Ammonia

Aluminum lactate treatment alters the lung biological activity of quartz.

Previous studies of surface modification of quartz particles have suggested that the biological activity of silica is at least in part related to its surface properties. In the present study, we exposed the tracheal lobe of 8 sheep to either 100 ml saline (saline group), 100 mg of quartz (Minusil-5) in 100 ml saline (SI group) or 100 mg of Al lactate treated quartz in 100 ml saline (SI-Al group). The 24 sheep were studied by bronchoalveolar lavage at days 0, 12, 24, 40, 60 and by autopsy at day 60. In the saline group, BAL analyses were as previously reported [1]. In the SI group, we found significant and sustained increases in total BAL cells (x 2, P less than 0.05), macrophages and lymphocytes (x 2, P less than 0.05), neutrophils (x 5-10, P less than 0.01), IgG (x 1.2-1.8, P less than 0.05), fibronectin (x 2-3, P less than 0.05), lactate dehydrogenase (x 3, P less than 0.01) and alkaline phosphatase (x 2, P less than 0.05). Histologically, a macrophagic and lymphocytic alveolitis was observed at day 60. In the SI-Al group, these changes were significantly attenuated and in the above parameters, SI-Al group did not differ from saline group after day 24. These data of BAL and histology of the sheep tracheal lobe model document clearly that aluminum lactate treatment alters the biological activity of quartz.

Animals

[Effect of neuraminidase and x-rays (2 Gy and 8 Gy) on microvilli and membrane invaginations of Ehrlich ascites tumor cells in monolayer culture].

A monolayer culture (Eagle basal medium plus 10% of fetal calf serum) of Ehrlich ascites tumor cells was exposed to X-radiation with 2 Gy and 8 Gy and treated with Vibrio cholerae neuraminidase alone or combined with sublethal X-ray irradiation (2 Gy). Pictures of the Ehrlich ascites tumor cells taken with the electron microscope were investigated in order to find out any cell surface modifications due to membrane invaginations and microvilli. The results showed that the rate of microvilli as well as that of membrane invaginations became higher with the increasing X-ray dose (2 Gy; 8 Gy). Following to neuraminidase treatment there was a considerable augmentation of membrane invaginations as compared to control cells, whereas the number of microvilli was slightly reduced. As it has been already described before, the influence of neuraminidase produced an increased endocytosis activity and a strengthening of the cytoskeleton. Combined treatment with neuraminidase and sublethal X-radiation (2 Gy) caused a higher rate of membrane invaginations than each method alone; the number of microvilli was slightly increased by combined treatment. The conclusion is drawn that these structure modifications are due to reparation processes induced by radiation on the one hand and to an enzymic action of neuraminidase on the cell surface on the other hand.

Animals

Plaque formation in vivo and bacterial attachment in vitro on permanently hydrophobic and hydrophilic surfaces.

Highly hydrated polyethylene oxide (PEO) films represent one type of surface modification which may interfere with biofilm formation. Protein adsorption and saliva-mediated bacterial adherence were investigated in vitro on normal and hydrophobized glass surfaces and on glass surfaces with immobilized PEO films. More protein and bacteria bound to untreated compared to hydrophobized and PEO-treated glass. Pellicle and plaque formation was also studied in vivo on ceramic crown surfaces either untreated, hydrophobized or with immobilized PEO films. Pellicle and plaque formation was similar on the untreated ceramic and PEO surfaces. Less plaque seemed to collect on these surfaces compared to adjacent normal tooth surfaces. Almost no plaque accumulated on the hydrophobic crown surface and it was virtually devoid of stainable pellicle. Even after 7 days in the mouth without oral hygiene this surface was very hydrophobic and the disclosing solution could not spread.

Actinomyces

The effect of chemical modification of quartz surfaces on particulate-induced pulmonary inflammation and fibrosis in the mouse.

One of the critical steps in the development of crystal-induced lung diseases is thought to be the interaction of crystal surfaces with cell membranes. The effect of chemical modifications of the surface of alpha-quartz on the development of lung disease has been investigated by treating quartz with various organosilanes. The functional groups attached to the quartz surfaces were (-CN), (-CH3), (-NH2), and -(N(CH3)3+). After intratracheal injection of each modified crystal at a constant surface area into mice, pulmonary inflammation and fibrosis were assessed 6 wk postexposure to the crystals by lung wet weight (lung index) and by the level of hydroxyproline in the lung. The crystals showing the highest degree of biologic activity were native quartz, which has a negative charge, -N(CH3)3+ modified quartz, which has a positive charge, and -CN modified quartz, which has no charge. One of the crystals with chemical groups capable of hydrogen bonding, the -NH2 modified quartz, was as unreactive as the crystal preparation modified with a hydrophobic group, -CH3. If the -CH3 and -NH2 modified quartz are compared as a less reactive group with the more reactive native quartz and -N(CH3)3+ modified quartz, these experiments suggest that electrostatic interactions may be more important in determining effective biologic activities than are hydrogen bonding interactions.

Animals

Spectroscopic characterization of polyethyleneglycol modified superoxide dismutase: 1H NMR studies on its Cu2Co2 derivative.

Spectroscopic methods have been employed in order to understand the molecular basis of the decrease in enzymatic activity of the antiinflammatory enzyme copper-zinc superoxide dismutase (SOD) following the covalent binding of polyethyleneglycol (PEG) chains to the protein amino-groups. The PEG modification is a general method recently proposed to improve the therapeutic index of enzymes. 1H NMR spectra on the cobalt substituted PEG-modified SOD, Cu2Co2-PEG-SOD, have been recorded. The signals are quite broad with respect to the unmodified enzyme. This has been interpreted on the basis of the effect of molecular weight on the linewidth. The analysis has shown that the histidine hydrogens involved in metal binding at the enzyme active site are the same in both native and PEG-modified SOD. Similarly, circular dichroism and absorption spectra indicate that the overall conformation of the metal clusters is not perturbed upon modification. On the other hand, azide titration shows that the affinity constant of N-3 for SOD is largely reduced upon PEG modification (K = 154 M-1 and 75 M-1 for the native and modified SOD, respectively). These results indicate that the decrease in enzymatic activity upon surface modification with PEG is not caused by a perturbation of the active site geometry, but to a decrease in the channeling of the O2- ion towards the enzyme active site.

Circular Dichroism

ESCA study on dental alloy surfaces modified by Ga-Sn alloy.

A new, simple surface modification method for adherend metals has been developed. It gives high bond strength and superior water durability to dental precious-metal alloys bonded with 4-META/MMA-TBB resin. However, there was no effect on the bonding of Ag-In-Zn alloy and base-metal alloys. In the present study, the alloy surfaces modified by the new method were analyzed by ESCA and SEM for determination of details of the modification effect. A new alloying layer containing Ga and Sn was formed on the precious-metal alloys. The main factor for excellent adhesion to be achieved was the formation of a very thin layer of Ga2O3 and SnO2, less than 1-2nm thick, on the alloy surface. A thicker modified layer, as formed on the Ag-In-Zn and Ni-Cr alloys, led to low bonding ability.

Acrylic Resins

Post-testicular change in the reptile sperm surface with particular reference to the snake, Natrix fasciata.

Sperm surface changes occurring in the reptile Wolffian duct have been explored with particular references to the snake, Natrix fasciata. In the snake Wolffian duct there are several proteins not present in serum, the pattern of which changes in concert with the seasonal testicular cycle. Whereas testicular spermatozoa did not bind antibody to duct secretions, all Wolffian duct spermatozoa did so over both head and tail, according to immunofluorescence patterns. Thus, on entering the Wolffian duct, the entire surface of N. fasciata spermatozoa acquires one of more of the duct's secretory components. As indicated by immunofluorescence, immunoelectrophoresis, and immunodiffusion, epitopes on at least some molecules that bind to spermatozoa or that remain free in the duct fluid are shared with those in other Natrix species, but not in more distant reptiles (turtle, anole lizard), nor chicken, rat, or rabbit. In regard to glycoproteins, one prominent con A-reactive band was present in polyacrylamide gel electrophoresis (PAGE) of snake fluid and more were evident in fluid collected from the turtle duct. However, such lectin-reactive elements did not bind to spermatozoa as judged by an absence of any change in snake, turtle and lizard sperm lectin-binding patterns in passing from the testis into and through the Wolffian duct. In all, evidence from these and other species studied begins to suggest that the nature of the post-testicular sperm surface modification displayed in most vertebrates that fertilize internally may differ in sub-therian and therian groups, respectively. There appears to be a relative emphasis on glycosyl-rich surface elements in the latter. The possible significance of these changes for sperm function in the different groups is discussed briefly in terms of sperm survival/storage, as well as capacitation and sperm binding to the zona.

Animals