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NIH workshop report. Biomaterials and medical implant science: present and future perspectives
Eisenberger, P. and D. Rekow (1997), Asaio J 43(1): 95-103.

NIST workshop on needs for reference biomaterials
Tesk, J. A. (2000), J Biomed Mater Res 51(2): 155-6.

Nitric oxide inhibition of platelet deposition on biomaterials
Ramamurthi, A. and R. S. Lewis (1999), Biomed Sci Instrum 35: 333-8.
Abstract: Platelet adhesion and aggregation restrict the clinical applicability of blood-contacting biomaterials. Nitric oxide (NO) is a simple biological molecule that may be incorporated into biomaterials to inhibit platelet deposition. The toxicity of NO at superphysiological levels necessitates the determination of aqueous NO concentrations and fluxes that effectively inhibit platelet deposition. In this study, a novel NO delivery device has been developed to study NO inhibition of platelet deposition in a dynamic in vitro environment. Gaseous NO was delivered via a semipermeable membrane to a radiolabeled platelet suspension perfusing a thin flow slit. The membrane was coated with a platelet-agonistic protein. Spatial NO flux and concentration profiles in the flow slit are predictable using a mathematical model. Platelet inhibition was essentially complete at 0.1 ppm gaseous NO exposure, corresponding to a surface concentration of 0.09 nM and surface fluxes between 0.3 and 0.6 femtomoles cm-2s-1. These threshold values of NO exposure for significant platelet inhibition were unchanged irrespective of the platelet agonist, perfusion times, or shear rates. At lower NO exposures (0.02 ppm), platelet inhibition was only partial with the degree of inhibition dependent on the nature of the agonistic protein. This study yields information useful towards the design and development of biomaterials incorporating NO for the reduction of platelet-biomaterial interactions.

Nitric oxide-eluting polyurethanes--vascular grafts of the future?
Verma, S. and P. A. Marsden (2005), N Engl J Med 353(7): 730-1.

Nitric oxide-producing polyurethanes
Jun, H. W., L. J. Taite, et al. (2005), Biomacromolecules 6(2): 838-44.
Abstract: Thrombus formation and eventual intimal hyperplasia are the leading causes of small-diameter synthetic vascular graft failure. To combat these issues, we have incorporated a diazeniumdiolate-modified nitric oxide (NO)-producing peptide into a polyurethane to improve the thromboresistance of this biocompatible polymer. NO production by polyurethane films occurred for approximately 2 months under physiological conditions, and mechanical properties of the material were suitable for vascular graft applications. Platelet adhesion to NO-releasing polyurethane was dramatically decreased compared to control polyurethane. Furthermore, endothelial cell growth was stimulated in the presence of the NO-releasing polyurethane, while smooth muscle cell growth was greatly inhibited. The ability of this bioactive material to inhibit platelet adhesion and smooth muscle cell proliferation while encouraging endothelialization suggests that this NO-generating polyurethane may be suitable as a candidate material for small-diameter vascular grafts.

Nitrite-modified extracellular matrix proteins deleteriously affect retinal pigment epithelial cell function and viability: a comparison study with nonenzymatic glycation mechanisms
Wang, Z., D. C. Paik, et al. (2005), Curr Eye Res 30(8): 691-702.
Abstract: PURPOSE: Extracellular matrix (ECM) plays an important role in the regulation of cell function. The aging process may involve chemical modifications to ECM proteins, which may contribute to the aging of the Bruch membrane and pathogenesis of age-related macular degeneration (AMD). The purpose of this study is to investigate nitrite modification of basement membrane-like proteins on RPE cell behavior as a model for the aging of the Bruch membrane in age-related eye diseases. As a comparison, retinal pigment epithelium (RPE) cell behavior on glycolaldehyde-modified matrices (GMM) was also studied. METHODS: Growth factor reduced Matrigel was reacted with nitrite or glycolaldehyde for 1 week or 12 hr, respectively. Calf RPE cells were plated on the modified matrices and examined in several ways. Attachment rates, proliferation rates, apoptosis, and necrosis were determined. Cell morphology and cell susceptibility to A2E-mediated damage was also monitored. RESULTS: Nitrite-modified matrices (NMMs) inhibited cell attachment by 65% and proliferation by 33.7% compared to 69.6% and 21.7%, respectively, by GMM. Proliferation inhibition was not significant when cells were plated at high density on GMM (3.47%) but significant on NMM (20.9%). NMM induced cell apoptosis and necrosis, but GMM induced cell apoptosis only. Both modifications inhibited RPE differentiation. RPE cells on both matrices were more susceptible to blue light mediated damage by A2E, but damage was greater on NMM. CONCLUSIONS: NMM has significant damaging effects on RPE cell function and viability that is similar to the damaging effects of GMM. These studies may have relevance to the RPE dysfunction observed during the progression of AMD.

NMR spectroscopy of proteins encapsulated in a positively charged surfactant
Lefebvre, B. G., W. Liu, et al. (2005), J Magn Reson 175(1): 158-62.
Abstract: Traditionally, large proteins, aggregation-prone proteins, and membrane proteins have been difficult to examine by modern multinuclear and multidimensional solution NMR spectroscopy. A major limitation presented by these protein systems is that their slow molecular reorientation compromises many aspects of the more powerful solution NMR methods. Several approaches have emerged to deal with the various spectroscopic difficulties arising from slow molecular reorientation. One of these takes the approach of actively seeking to increase the effective rate of molecular reorientation by encapsulating the protein of interest within the protective shell of a reverse micelle and dissolving the resulting particle in a low viscosity fluid. Since the encapsulation is largely driven by electrostatic interactions, the preparation of samples of acidic proteins suitable for NMR spectroscopy has been problematic owing to the paucity of suitable cationic surfactants. Here, it is shown that the cationic surfactant CTAB may be used to prepare samples of encapsulated anionic proteins dissolved in low viscosity solvents. In a more subtle application, it is further shown that this surfactant can be employed to encapsulate a highly basic protein, which is completely denatured upon encapsulation using an anionic surfactant.

No effect of a type I collagen gel coating in uncemented implant fixation
Svehla, M., P. Morberg, et al. (2005), J Biomed Mater Res B Appl Biomater 74(1): 423-8.
Abstract: Uncemented joint replacement with a variety of substrate materials, structures, and coatings are commonplace in arthroplasty. Even with specialized surgical preparation of bone, intimate contact between the implant and host bone may not always be achieved. This study evaluated the in vivo effect of fibrillar atelopeptide and PEG crosslinked collagens coatings placed directly into porous sintered bead structures on bone ingrowth using a skeletally mature bicortical, bilateral ovine tibia model. Bone ingrowth into the implants increased with time, although differences were not significant. At 4 weeks woven bone was present within the pores that remodeled with time. Significantly lower levels of ingrowth were observed in the intramedullary region of the implants when compared with the cortical region. Implant metal type did not affect ingrowth in both regions analyzed. Both fibrillar and crosslinked forms of dermal type I collagen did not significantly alter bone ingrowth.

No effect of hydroxyapatite particles in phagocytosable sizes on implant fixation: an experimental study in dogs
Rahbek, O., S. Kold, et al. (2005), J Biomed Mater Res A 73(2): 150-7.
Abstract: The influence of wear debris on bone healing around orthopedic implants is debated. Hydroxyapatite (HA) particles and polyethylene (PE) particles have been shown to have a negative effect on osteoblast cultures in vitro. The present study investigated the in vivo effects of HA and PE particles on the mechanical fixation and gap healing around experimental HA implants. Nonloaded implants (n = 30) were inserted bilaterally into the proximal tibia of 15 dogs with a 2-mm gap to the bone. The peri-implant gap was either (1) empty (n = 6) or filled with (2) hyaluronic acid (n = 8), (3) hyaluronic acid and HA particles (n = 8), or (4) hyaluronic acid and PE particles (n = 8). After 4 weeks, the animals were killed. The implant interface was evaluated by pushout testing until failure and by histomorphometry. Both HA and PE particles were found to be phagocytosed by macrophage-like cells in the interfacial tissue. HA particles were also integrated in newly formed bone. We found no negative effect of the particulate material on mechanical fixation of the implants or on bone formation around the implants.

No negative effects of bone impaction grafting with bone and ceramic mixtures
Arts, J. J., J. W. Gardeniers, et al. (2005), Clin Orthop Relat Res 438: 239-47.
Abstract: Reconstructing large loaded bone defects with ceramic bone graft extenders is tempting considering the expected future donor bone shortage. However, whether there are negative effects is unknown. Standardized large defects in the acetabulum of goats were created and subsequently reconstructed with metal mesh and impacted morselized cancellous bone grafts or a 50/50% volume mixture of tricalcium phosphate-hydroxyapatite granules and morselized cancellous bone grafts using the bone impaction grafting technique. Subsequently, a cemented total hip prosthesis was inserted. Clinically, no differences were observed between groups. Most of the morselized cancellous bone graft had been resorbed and incorporated into new bone after 15 weeks. The large tricalcium phosphate-hydroxyapatite granules were integrated, the smaller crushed tricalcium phosphate-hydroxyapatite granules were surrounded by osteoclasts or engulfed by macrophages and giant cells. The cement penetration into the reconstructive layer and the quality of the bone based on a semiquantitative score were similar in both groups. We found no suggestion of tricalcium-hydroxyapatite granule-induced third-body wear in this short-term followup study. No negative effects were observed in this study, and therefore, it seems reasonable to use tricalcium-hydroxyapatite granules in a 50/50% volume mix with morselized cancellous bone graft as a bone graft extender in acetabular revision surgery with the bone impaction grafting technique.

Noggin and bFGF cooperate to maintain the pluripotency of human embryonic stem cells in the absence of feeder layers
Wang, G., H. Zhang, et al. (2005), Biochem Biophys Res Commun 330(3): 934-42.
Abstract: Human embryonic stem (hES) cells are typically maintained on mouse embryonic fibroblast (MEF) feeders or with MEF-conditioned medium. However, these xenosupport systems greatly limit the therapeutic applications of hES cells because of the risk of cross-transfer of animal pathogens. Here we showed that the bone morphogenetic protein antagonist noggin is critical in preventing differentiation of hES cells in culture. Furthermore, we found that the combination of noggin and basic fibroblast growth factor (bFGF) was sufficient to maintain the prolonged growth of hES cells while retaining all hES cell features. Since both noggin and bFGF are expressed in MEF, our findings suggest that they may be important factors secreted by MEF for maintaining undifferentiated pluripotent hES cells. Our data provide new insight into the mechanism how hES cell self-renewal is regulated. The newly developed feeder-free culture system will provide a more reliable alternative for future therapeutic applications of hES cells.

Nonalloyed titanium as a bioinert metal--a review
Tschernitschek, H., L. Borchers, et al. (2005), Quintessence Int 36(7-8): 523-30.
Abstract: Titanium is used for many dental applications and instruments, such as orthodontic wires, endodontic files, dental implants, and cast restorations. The popularity of titanium is primarily due to its good mechanical properties, its high corrosion resistance, and its excellent biocompatibility. A thorough review of the medical and dental literature reveals, however, that titanium can also cause chemical-biological interactions. Tissue discoloration and allergic reactions in patients who have come in contact with titanium have been reported. The biostability of titanium is becoming increasingly questioned. At the same time, new technologies and materials, such as high-performance ceramics, are emerging which could replace titanium in dentistry in the not-too-distant future.

Noncovalent coatings for the separation of synthetic polypeptides by nonaqueous capillary electrophoresis
Vayaboury, W., D. Kirby, et al. (2005), Electrophoresis 26(11): 2187-97.
Abstract: Recently, we demonstrated the possibility to extend the range of capillary electrophoresis (CE) applications to the separation of non-water-soluble synthetic polymers. This work focuses on the control of the electro-osmotic flow (EOF) and on the limitation of the solute adsorption in nonaqueous electrolytes. For these purposes, different strategies were investigated. For the initial, a viscous additive (ethylene glycol or glycerol) was used in the electrolyte in order to decrease the EOF magnitude and, possibly, to compete with solute adsorption. A second strategy was to modify, before separation, the fused-silica capillary wall by the adsorption of poly(ethylene oxide) (PEO) via hydrogen bonding. The influence of the molecular mass of the adsorbed PEO on the EOF magnitude and direction was studied in electrolytes based on methanol/acetonitrile mixtures containing ammonium ions. For PEO molecular masses above 1000 g/mol, reversed (anodic) EOF were reported in accordance with previous results obtained with PEO covalently bonded capillaries. The influence of the nature and the concentration of the background electrolyte cation on the EOF magnitude and direction were also investigated. A third strategy consisted in modifying the capillary wall by the adsorption of a cationic polyelectrolyte layer. Advantageously, this polyelectrolyte layer suppressed the adsorption of the polymer solutes onto the capillary wall. The results obtained in this work confirm the high potential and the versatility of CE for the characterization of ionizable organic polymers in nonaqueous media.

Noncovalently bilayer-coated capillaries for efficient and reproducible analysis of proteins by capillary electrophoresis
Catai, J. R., H. A. Tervahauta, et al. (2005), J Chromatogr A 1083(1-2): 185-92.
Abstract: The suitability of noncovalently bilayer-coated capillaries for the analysis of proteins by capillary electrophoresis (CE) at medium pH was investigated. Fused-silica capillaries were coated simply by successively flushing with a polybrene (PB) and a poly(vinyl sulfonate) (PVS) solution. A protein test mixture was used to evaluate the performance of the coated capillaries. Comparisons with bare fused-silica capillaries were made. Several background electrolytes (BGEs) were tested in combination with the PB-PVS coating, showing that optimum performance was obtained for the proteins using high BGE concentrations. With a 300 mM Tris phosphate buffer (pH 7.0), good plate numbers (150,000-300,000), symmetrical peaks, and favorable migration-time repeatabilities (RSDs below 0.8%) were obtained for the proteins. Using bare fused-silica capillaries, the protein peaks were significantly broadened and the migration-time RSDs often exceeded 5%. It is concluded that the PB-PVS coating effectively minimizes adverse protein adsorption and provides a very stable electroosmotic flow (EOF). We also investigated the potential of a commercially available bilayer coating (CEofix) for protein analysis. It is demonstrated that with this coating, good plate numbers and peak symmetries for proteins can be achieved when the CEofix BGE ("accelerator") is replaced by a common BGE such as sodium or Tris phosphate. Apparently, the negatively charged polymer present in the "accelerator" interacts with the proteins causing band broadening. The utility of the bilayer coatings is further illustrated by the separation of proteins such as interferon-alpha 2b, myoglobin and carbonic anhydrase, by the analysis of a degraded insulin sample in time, and by the profiling of the glycoprotein ovalbumin. In addition, it is demonstrated that even in the presence of concentrations of human serum albumin in the sample of up to 60 mg/mL, the PB-PVS coating still provides reproducible protein separations of good performance.

Noninvasive bone replacement with a new injectable calcium phosphate biomaterial
Gauthier, O., I. Khairoun, et al. (2003), J Biomed Mater Res A 66(1): 47-54.
Abstract: The use of injectable calcium phosphate (CaP) biomaterials in noninvasive surgery should provide efficient bone colonization and implantation. Two different kinds of injectable biomaterials are presently under development: ionic hydraulic bone cements that harden in vivo after injection, and an association of biphasic calcium phosphate (BCP) ceramic granules and a water-soluble polymer vehicle (a technique particularly investigated by our group), providing an injectable CaP bone substitute (IBS). In our study, we compared these two approaches, using physicochemical characterizations and in vivo evaluations in light microscopy, scanning electron microscopy, and three-dimensional microtomography with synchrotron technology. Three weeks after implantation in rabbit bone, both biomaterials showed perfect biocompatibility and bioactivity, but new bone formation and degradation of the biomaterial were significantly greater for BCP granules than for ionic cement. Newly formed bone developed, binding the BCP granules together, whereas new bone grew only on the surface of the cement, which remained dense, with no obvious degradation 3 weeks after implantation. This study confirms that BCP granules carried by a cellulosic polymer conserve bioactivity and are conducive to earlier and more extensive bone substitution than a carbonated-hydroxyapatite bone cement. The presence of intergranular spaces in the BCP preparation, as shown on microtomography imaging, seems particularly favorable, allowing body fluids to reach each BCP granule immediately after implantation. Thus, the IBS functions as a completely interconnected ceramic with total open macroporosity. This new bone replacement approach should facilitate microinvasive bone surgery and local delivery of bone therapy agents.

Non-invasive magnetic resonance imaging of the soft tissue response to a biomaterial
Khor, E., J. A. Hunt, et al. (1993), Clin Mater 12(2): 65-72.
Abstract: Magnetic resonance imaging (MRI) has been employed to visualize the tissue response to hydrogel implants in rats. High contrast MR images of the implant site were obtained. Distinct tissue variations in the MR images have been observed. These can be attributed to either the surgical procedure or the application of a tissue irritant to produce inflammation and have been verified histologically. This study demonstrates that MRI is potentially a useful tool for the non-invasive in-vivo evaluation of biomaterials.

Non-viral-mediated gene therapy approaches for bone repair
Winn, S. R., J. C. Chen, et al. (2005), Orthod Craniofac Res 8(3): 183-90.
Abstract: OBJECTIVES: Bone repair strategies continue to be developed for alternatives to autografting, allogeneic implants of banked bone, and other bone substitutes. Efforts have included the delivery of potent growth and/or differentiation factors and the use of gene therapy. For bone regeneration, gene therapy is the delivery, uptake and expression of DNA that has been localized to a wound bed. The objective of the current study is to investigate methods to enhance non-viral-mediated means of gene uptake and expression for use in bone regeneration. METHODS: Several types of DNA-polymer complexes, either applied directly to baby hamster kidney (BHK) cells, or released from a porous, resorbable gene-activated matrix (GAM), were evaluated in vitro for their ability to transfect cells with a circular plasmid DNA construct expressing green fluorescent protein. Complexes included conjugates containing a lipophilic reagent, liposomes, poly-ethyl-oxazoline, and poly-ethyleneimine (PEI). Data were subjected to analysis of variance and Fisher's protected least significant difference for multiple comparisons with significance established at p < 0.05. RESULTS: Transfection efficiencies of the liposome and PEI complexes improved in vitro when released from resorbable GAMs. The lipophilic reagent FuGene 6 demonstrated abundant uptake and expression in the initial 1- and 2-day evaluation periods. In contrast, the DNA-liposome and PEI GAM complexes demonstrated a sustained release, uptake and expression by the BHK cells at the 2-, 4-, and 7-day, and 4- and 7-day evaluation intervals, respectively. CONCLUSION: GAM technology appears to improve the functional stability and release duration of incorporated DNA-polymer complexes in the present in vitro studies. The ongoing objective of our research is to develop a localized treatment to improve the uptake and expression of plasmid DNA by non-viral-mediated gene therapy.

Novel acrylonitrile-based copolymers containing phospholipid moieties: synthesis and characterization
Huang, X. J., Z. K. Xu, et al. (2005), Macromol Biosci 5(4): 322-30.
Abstract: Novel acrylonitrile-based copolymers containing phospholipid moieties were synthesized by a three-step process, which included the copolymerization of acrylonitrile and 2-hydroxyethyl methacrylate (HEMA) in water and the reaction of the resulting poly[acrylonitrile-co-(2-hydroxyethyl methacrylate)]s (PANCHEMA) with 2-chloro-2-oxo-1,3,2-dioxaphospholane (COP) followed by the ring-opening reaction of COP with trimethylamine. The chemical structure of PANCHEMA and the phospholipid-containing acrylonitrile-based copolymers (PLCANCP) was analyzed with FT-IR spectroscopy, (1)H and (31)P NMR, and XPS. Surface properties of the studied copolymers were evaluated by the pure water contact angle, protein adsorption and platelets adhesion measurements. The water contact angle measured by sessile drop method decreased for the polymers in the following sequence: PAN, PANCHEMA, and PLCANCP. The adsorption amount of bovine serum albumin and the adhesive number of platelet followed the same decline sequence. These results demonstrate that the biocompatibility of the acrylonitrile-based copolymer membranes could be improved efficiently by the introduction of phospholipid moieties.

Novel alginate-poly(L-histidine) microcapsules as drug carriers: in vitro protein release and short term stability
Wang, S. B., F. H. Xu, et al. (2005), Macromol Biosci 5(5): 408-14.
Abstract: Spherical, smooth-surfaced and mechanically stable alginate-poly(L-histidine) (PLHis) microcapsules with narrow particle size distributions were prepared by incubating calcium alginate beads in aqueous solutions of PLHis. The in vitro release characteristics, drug loading and encapsulation efficiency of the microcapsules were investigated using bovine erythrocytes hemoglobin (Hb) as a model drug. The results showed that the concentration of Ca(2+) ions had a considerable effect on the drug loading, encapsulation efficiency and in vitro release behavior of the microcapsules. When the concentration of CaCl(2) in the PLHis solution was increased from 0 to 3.0% (w/v), the drug loading and encapsulation efficiency decreased significantly from 38.0 to 4.3% and from 92.9 to 8.0%, respectively, while the total cumulative release of Hb from microcapsules in phosphate buffered saline solution (PBS, pH 6.8) decreased from 96.2 to 72.8% in 24 h. No significant protein release was observed during 70 h of incubation in hydrochloric acid solution (pH 1.2). However, under neutral conditions (PBS, pH 6.8), the Hb was completely and stably released within 24-70 h. An explosion test showed that the stability of alginate-PLHis microcapsules depended strongly on the concentration of PLHis and the calcium ions in solution. [Diagram: see text] Microscopy photo of Hb-loaded alginate-PLHis microcapsules.

Novel approach for quantification of porosity for biomaterial implants using microcomputed tomography (microCT)
Hiu-Yan, Y., Q. Ling, et al. (2005), J Biomed Mater Res B Appl Biomater 75(2): 234-42.
Abstract: Porous bioceramics have been widely investigated in orthopaedic tissue engineering. Attention has been given to manufacturing of a porous bioceramic that mimics the trabecular bone structure for proper bone regeneration. With the advance of biomedical imaging through microcomputed tomography (microCT), this study attempted to quantify the pore structure of different bioceramics. Two bioceramic blocks (BSC and ChronOS) were synthesized by two methods. The specification claimed the porosity of the bioceramic ranged from 40% to 70%. Six blocks of each bioceramic were evaluated by conventional water immersion method and microCT. The pore size and connectivity were evaluated with standardized protocols. By the water immersion method, the porosity of BSC and ChronOS was 60.4% and 74.7%, respectively. The three-dimensional results of microCT showed that BSC porosity was 26.2% and ChronOS was 60.0%. The pore connectivity was evaluated to be 2.6 for BSC and 39.7 for ChronOS. ChronOS had functional pores with 200 microm to 400 microm in diameter (87.8%+/-0.5%), which is significantly more than 52.8%+/-11.5% of pores in BSC (p<0.05). Providing information on the functional pores objectively, the microCT evaluation serves as a good standard for specification of the bioceramic-related implants.

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