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Human monocyte/macrophage adhesion, macrophage motility, and IL-4-induced foreign body giant cell formation on silane-modified surfaces in vitro. Student Research Award in the Master's Degree Candidate Category, 24th Annual Meeting of the Society for Biomaterials, San Diego, CA, April 22-26, 1998
Jenney, C. R., K. M. DeFife, et al. (1998), J Biomed Mater Res 41(2): 171-84.
Abstract: A cytokine-based, in vitro model of foreign body giant cell (FBGC) formation was utilized to examine the effect of biomaterial surface chemistry on the adhesion, motility, and fusion of monocytes and macrophages. Human monocytes were cultured for 10 days on 14 different silane-modified glass surfaces, during which time the cells assumed the macrophage phenotype. The adhesion of monocytes and macrophages during the culture period decreased by an average of approximately 50%, with the majority of cell loss observed during days 1-3. Most important, the adhesion of monocytes and macrophages was surface independent except for two surfaces containing terminal methyl groups, which decreased adhesion levels. Interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were added to the medium to induce FBGC formation and enhance macrophage adhesion, respectively. Surprisingly, GM-CSF decreased long-term monocyte/macrophage adhesion. IL-4-induced FBGC density was strongly influenced by the surface carbon content, as determined by X-ray photoelectron spectroscopy (XPS). In contrast, contact angle and surface energy displayed no correlation with FBGC formation. The motility of adherent macrophages, as measured by time-lapse confocal microscopy, was not affected significantly by differences in surface chemistry or the addition of cytokines. The surface dependence of FBGC formation is hypothesized to be the result of varying levels of silane-derived surface carbon.

Human monocyte-derived macrophages and dendritic cells as targets for biomaterial cytocompatibility studies using an improved in vitro culture system
Mebouta-Nkamgueu, E., J. J. Adnet, et al. (2001), J Mater Sci Mater Med 12(4): 351-7.
Abstract: Since macrophage plays a key role in the biocompatibility process, neoplastic macrophage cell lines and human blood monocytes are commonly used as target cells for in vitro biomaterial tolerance evaluation. However, tumor cells profoundly differ from normal tissue cells and monocytes are only precursors of macrophages. It has become possible to generate recently, under adherent-free conditions, fully mature macrophages and dendritic cells from human blood monocytes in the presence of GM-CSF and GM-CSF + IL4 respectively. In the present work, we examined the effects of titanium-alloy on morphology, adhesion, cell phenotype and TNF-alpha release activity of such differentiated cells grown in hydrophobic teflon bags. Scanning electron microscopy showed that macrophages substantially adhered and spread on titanium-alloy surface throughout the culture period, whereas only a few dendritic cells were adherent. The phenotype of both cell types remained unchanged in the presence of the tested material. However, titanium-alloy stimulated the secretion of TNF-alpha by the macrophages of some donors. This model of culture may offer new insights into the biomaterial evaluation and may be useful for studying individual responses induced by biomaterials.

Human osteoblast in contact with various biomaterials in vitro
Kudelska-Mazur, D., M. Lewandowska-Szumiel, et al. (1999), Ann Transplant 4(3-4): 98-100.
Abstract: OBJECTIVES: The aim of the study was to investigate changes in osteoblast number and viability in contact with biomaterials and to compare both tests. METHODS: Human primary culture osteoblasts were seeded on the polished surfaces of hydroxyapatite, alumina, titanium and surgical steel. After 4, 9, 14, 24 and 48h cultures were subjected to XTT viability assay. Subsequently Hoechst staining of nuclei was performed. Number of cells on each sample was counted. RESULTS: There were no differences in cell viability measured by means of XTT assay between osteoblasts cultured on hydroxyapatite and alumina during 48 hours of experiment. However on titanium as well as on surgical steel cell viability was significantly lower than on bioceramics. The lowest viability was noticed on surgical steel. Cell number on titanium was significantly higher than on steel. There were no differences between cell numbers on hydroxyapatite and alumina as well as between investigated bioceramics and metals. Nucleus number and the results of viability assay were compared. There was no correlation found between number and viability of cells. CONCLUSION: the results of a single test may not provide sufficient information on the interaction between cells and implant. Application of a battery of tests is necessary in material biocompatibility investigation in vitro.

Human placental explants in culture: approaches and assessments
Miller, R. K., O. Genbacev, et al. (2005), Placenta 26(6): 439-48.
Abstract: Placental explant cultures in vitro are useful for studying tissue functions including cellular uptake, production and release of secretory components, cell interactions, proliferation, growth and differentiation, gene delivery, pharmacology, toxicology, and disease processes. A variety of culture conditions are required to mimic in utero environments at different times of gestation including differing oxygen partial pressures, extracellular matrices and culture medium. Optimization of explant methods is examined for first and third trimester human placental tissue and the biological processes under investigation.

Human serum opsonization of orthopedic biomaterial particles: protein-binding and monocyte/macrophage activation in vitro
Sun, D. H., M. C. Trindade, et al. (2003), J Biomed Mater Res A 65(2): 290-8.
Abstract: Wear particles generated after total joint arthroplasty activate monocyte/macrophages and incite formation of a granulomatous periprosthetic tissue associated with bone loss and implant loosening. This study tested the hypothesis that selective opsonization of orthopedic implant biomaterial wear particles by human serum proteins influences monocyte/macrophage activation. Serum protein binding to metallic, polymeric, and ceramic particles was determined by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Individual proteins bound to particles were subsequently identified using two-dimensional SDS-PAGE, microsequencing techniques, and SWISS-PROT analysis. Effects of selective protein opsonization on particle-induced monocyte/macrophage activation were assessed by quantification of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha release. Results from one-dimensional gel analyses revealed distinct serum protein-binding patterns specific for each material tested. Two-dimensional gel analysis together with amino acid sequencing of the prominent protein species confirmed the presence of albumin and alpha-1-antitrypsin bound to all particles tested. In contrast to the metallic particles, apolipoprotein was a major species associated with polymeric particles. Opsonization of PMMA particles with purified preparations of each of the identified proteins showed that albumin significantly enhanced particle-induced monocyte/macrophage activation. These data confirm orthopedic biomaterial specific binding of human serum proteins and demonstrate that albumin exacerbates particle-induced monocyte/macrophage activation. Alterations in the chemical and surface properties of orthopedic biomaterials to modulate protein interactions may improve implant longevity.

Human tissue scaffolds: measured progress ensures a safe future
Tomlins, P. (2005), Med Device Technol 16(5): 29-31.
Abstract: Measurement scientists are helping the tissue-engineering community develop robust manufacturing routes and a regulatory framework for tissue scaffolds. Activities are described that are underway to make tissue engineered products a commercial reality.

Human-serum matrix supports undifferentiated growth of human embryonic stem cells
Stojkovic, P., M. Lako, et al. (2005), Stem Cells 23(7): 895-902.
Abstract: One of the most frequently used matrices for feeder-free growth of undifferentiated human embryonic stem cells (hESCs) is Matrigel, which supports attachment and growth of undifferentiated hESCs in the presence of mouse embryonic fibroblast-conditioned medium. Unfortunately, application of Matrigel or medium conditioned by mouse embryonic feeder cells is not ideal for potential medical application of hESCs because xenogeneic pathogens can be transmitted through culture conditions. We demonstrate here that human serum as matrix and medium conditioned by differentiated hESCs reduce exposure of hESCs to animal ingredients and provide a safer direction toward completely animal-free conditions for application, handling, and understanding of hESC biology. At the same time, hESCs grown under these conditions maintain all hESC features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype.

Humoral immune responses to model antigen co-delivered with biomaterials used in tissue engineering
Matzelle, M. M. and J. E. Babensee (2004), Biomaterials 25(2): 295-304.
Abstract: A model shed antigen, ovalbumin (OVA), was co-delivered with polymeric biomaterial carrier vehicles in C57BL6 mice to test whether the presence of the biomaterial acted as an adjuvant in the immune response towards the associated antigen. The biomaterials tested were non-biodegradable polystyrene microparticles and biodegradable 50:50 or 75:25 poly(lactic-co-glycolic acid) (PLGA) microparticles or scaffolds. For each biomaterial carrier vehicle, to assess the resulting time-dependent systemic humoral immune response towards the co-delivered OVA, the OVA-specific IgG concentration and isotypes (IgG2a or IgG1, indicating a predominant Th1 or Th2 response, respectively) were determined using ELISA. OVA co-delivered with biomaterial carrier vehicles supported a moderate humoral immune response that was maintained for the 18-week duration of the experiment. This humoral immune response was primarily Th2 helper T cell-dependent as indicated by the predominant IgG1 isotype. Furthermore, this humoral immune response was not material chemistry-dependent within the material set tested here. With the presence of the biomaterial resulting in an enhancement of the humoral immune response to co-delivered antigen, it appears that the biomaterial acts as an adjuvant in the development of an adaptive immune response to co-delivered antigen.

Hyaluronan-based biomaterial (Hyaff-11) as scaffold to support mineralization of bone marrow stromal cells
Lisignoli, G., S. Toneguzzi, et al. (2003), Chir Organi Mov 88(4): 363-7.
Abstract: Various techniques are widely used to repair bone defects, association of hyaluronan-based biodegradable polymers (Hyaff-11) with bone marrow stromal cells (BMSC) promises to provide successful cell scaffolds for tissue-engineered repair of bone tissue. We evaluate in vitro and in vivo the potential of Hyaff-11 to facilitate mineralization of BMSC. Rat BMSC were seeded on Hyaff-11 and their differentiation were assessed at different time points. Osteogenic differentiation was investigated in vitro analysing the expression of alkaline phosphatase and osteocalcin. Mineralization of bone defects was evaluated also in vivo implanting Hyaff-11 scaffold combined with BMSC in large segmental radius defects. In vitro, we found a decrease expression of alkaline phosphatase and an increase of osteocalcin. In vivo, our data showed that mineralization was induced and basic fibroblast growth factor contributed to this process. These results provide a demonstration to therapeutic potential of Hyaff-11 as appropriate carrier vehicle for differentiation and mineralization of BMSC and for the repair of bone defects.

Hyaluronate-alginate combination for the preparation of new biomaterials: investigation of the behaviour in aqueous solutions
Oerther, S., E. Payan, et al. (1999), Biochim Biophys Acta 1426(1): 185-94.
Abstract: With the aim of producing a biomaterial for surgical applications, the alginate-hyaluronate association has been investigated. Crossed techniques were used to assess the existence of polymer interactions in aqueous solutions up to 20 mg/ml. Alginate was obtained from algae and hyaluronate was purified from rooster comb. Viscometry measurements using the capillary technique or the Couette flow, together with circular dichroism investigations, evidenced the moderate significance of interactions between the two polysaccharides in dilute solutions. In addition, the case of more concentrated solutions and containing 20 mg/ml alginate was approached by rheological measurements in the flow mode; the behaviour of the polymer associations appeared as a compromise between those of individual polysaccharides.

Hyaluronate-alginate gel as a novel biomaterial: mechanical properties and formation mechanism
Oerther, S., H. Le Gall, et al. (1999), Biotechnol Bioeng 63(2): 206-15.
Abstract: With the aim of producing a biomaterial for surgical applications, the alginate-hyaluronate association has been investigated to combine the gel-forming properties of alginate with the healing properties of hyaluronate. Gels were prepared by diffusion of calcium into alginate-hyaluronate mixtures, with an alginate content of 20 mg/mL. The hyaluronate source was shown to have significant effect on the aspect and the properties of the gels. The gels have viscoelastic behaviour and the transient measurements carried out in creep mode could be interpreted through a Kelvin-Voigt generalised model: experimental data led to the steady state hardness and a characteristic viscosity of the gel. Gels prepared from Na rooster comb hyaluronate with weight ratio up to 0.50 have satisfactory mechanical properties, and fully stable gels are obtained after a few days; on the contrary, use of lower molecular weight hyaluronate led to loose gels for hyaluronate contents over 0.25. Gel formation was investigated by measurements of the exchange fluxes between the calcium chloride solution and the forming gel, which allowed thorough investigations of the occuring diffusion phenomena of water, calcium ion and hyaluronate. Strong interactions of water with hyaluronate reduce significantly the rate of weight loss from the gel beads and allows higher water content in steady-state gels. Calcium content in the gel samples could be correlated to the actual alginate concentration, whatever the nature and the weight ratio of hyaluronate.

Hyaluronic acid-poly-D-lysine-based three-dimensional hydrogel for traumatic brain injury
Tian, W. M., S. P. Hou, et al. (2005), Tissue Eng 11(3-4): 513-25.
Abstract: Brain tissue engineering in the postinjury brain represents a promising option for cellular replacement and rescue, providing a cell scaffold for either transplanted or resident cells. In this article, a hyaluronic acid (HA)-poly-D-lysine (PDL) copolymer hydrogel with an open porous structure and viscoelastic properties similar to neural tissue has been developed for brain tissue engineering. The chemicophysical properties of the hydrogel with HA:PDL ratios of 10:1, 5:1, and 4:1 were investigated by scanning electron microscopy (SEM) and X-ray photoelectron spectrometry. Neural cells cultured in the hydrogel were studied by phase-contrast microscope and SEM. The incorporation of PDL peptides into the HA-PDL hydrogel allowed for the modulation of neuronal cell adhesion and neural network formation. Macrophages and multinucleated foreign body giant cells found at the site of implantation of the hydrogel in the rat brain within the first weeks postimplantation decreased in numbers after 6 weeks, consistent with the host response to inert implants in numerous tissues. Of importance was the infiltration of the hydrogel by glial fibrillary acidic protein-positive cells-reactive astrocytes-by immunohistochemistry and the contiguity between the hydrogel and the surrounding tissue demonstrated by SEM. These findings indicated the compatibility of this hydrogel with brain tissue. Collectively, the results demonstrate the promise of an HA-PDL hydrogel as a scaffold material for the repair of defects in the brain.

Hyaluronidase-bound membrane as a biomaterial for implantable fuel cells
Ahn, B. K., S. K. Wolfson, Jr., et al. (1976), J Biomed Mater Res 10(2): 283-94.
Abstract: A new biomaterial containing covalently bound hyaluronidase was prepared. An application of this enzyme membrane is to improve the performance of an implantable fuel cell. Hyaluronic acid is a contributor to the viscosity of tissue fluids but can be a potential fuel source because of its sugar content. The incorporation of immobilized hyaluronidase would not only contribute to a more available fuel supply by splitting hyaluronic acid but, perhaps more importantly, enhance the rate of mass transport of fuel, O2, and reaction products by reducing the viscosity near the electrode membranes. Hyaluronidase was bound to Sepharose gel and its thermoplastic membrane after activation by cyanogen bromide. Fourteen and 22% of the activities were recovered from the gel and membrane, respectively. The activity of the bound enzyme was stable for six months at 0 degrees C. The addition of hyaluronic acid, 1 mg/ml, to a typical implantable type bioautofuel cell in vitro increased external solution viscosity from 1.1 to 2.5-2.8 cP and reduced voltage output under 10 komega by 60% in 3 hr. When the hyaluronidase bound membrane was placed at the anode, viscosity of the glucose-hyaluronic acid solution was lowered to 1.8 cP and the cell output increased to the original level of a glucose-fueled cell in 3 hr. Glucosamine-equivalent released from hyaluronic acid at the electrode was 3.1 mg after 22.5 hr. This represents 90% of the theoretical consumption. Restoration of the cell output was probably a combination of the enhanced transport of fuel, O2 and products, and/or appearance of a new fuel, glucosamine-equivalent.

Hybrid biomaterials based on the interaction of polyurethane oligomers with porcine pericardium
Loke, W. K., E. Khor, et al. (1996), Biomaterials 17(22): 2163-72.
Abstract: Hybrid biomaterials have been produced by the interaction of polyurethane oligomers with both fresh and glutaraldehyde-fixed porcine pericardium. The hybrid biomaterials so formed were translucent with occasional white streaks and/or spots, had increased stiffness (to touch) but remained pliable. No shrinkage temperature was detected for fresh porcine pericardium hybrid up to 100 degrees C compared to porcine pericardium (approximately 67 degrees C) and glutaraldehyde-fixed porcine pericardium (approximately 87 degrees C). Amino acid analysis of the fresh porcine pericardium hybrid showed a reduction in lysine content after active isocyanate-terminated polyurethane oligomers exposure, indicating cross-linking between the polymer and tissue. Histological examination of the hybrid material shows a thin grey coating on both surfaces of the tissue, implying at least surface cross-linking of the tissue with polyurethane. The results suggest that fresh porcine pericardium can be reacted with active isocyanate-terminated polyurethane oligomers to produce hybrid biomaterials with covalent bonding.

Hybrid films from blends of chitosan and egg phosphatidylcholine for localized delivery of paclitaxel
Grant, J., M. Blicker, et al. (2005), J Pharm Sci 94(7): 1512-27.
Abstract: Chitosan and egg phosphatidylcholine (ePC) were used as a unique combination to prepare composite films for localized drug delivery. In comparison to other phospholipids analyzed, ePC was found to produce chitosan-based films with minimal swelling and a high degree of stability. The properties of the chitosan-ePC films were characterized and found to be dependent on the ratio of chitosan:ePC present. FTIR analysis of chitosan-ePC films revealed that their high stability may be attributed to interactions present between these two biomaterials. In vitro evaluation of the cytotoxicity and protein adsorption properties of the films were used to provide a preliminary indication of their biocompatibility. The chitosan-ePC film was also evaluated as a matrix for the localized delivery of the anti-cancer agent, paclitaxel. Nanoparticles containing paclitaxel were dispersed throughout the chitosan-ePC film to result in a drug:material ratio of 1:8 (wt/wt). The film was found to provide a sustained release of paclitaxel over a 4-month period in biologically relevant media. The biological activity of paclitaxel loaded in the chitosan-ePC film was confirmed in SKOV-3 human ovarian cancer cells.

Hybrid polysaccharide-silica nanocomposites prepared by the sol-gel technique
Shchipunov, Y. A. and T. Y. Karpenko (2004), Langmuir 20(10): 3882-7.
Abstract: New monolithic nanocomposite silica biomaterials were synthesized on the basis of various natural polysaccharides and recently introduced completely water-soluble precursor tetrakis(2-hydroxyethyl) orthosilicate. The sol-gel processes were performed in aqueous solutions without the addition of organic solvents and catalysts. The silica polymerization was promoted by the polysaccharides through acceleration and catalytic effect on the processes. By introducing poly(vinyl alcohol) or poly(ethylene oxide) in the precursor solution, it was shown that the jellification took place in the case of the hydroxyl-containing polymer. Therefore, it was suggested that the catalysis was caused by a formation of hydrogen bonds between hydroxyl groups in macromolecules and products of precursor hydrolysis (silanols). It was also demonstrated that the polysaccharides radically changed the structure of biomaterials. In place of the cross-linked nanoparticles, there was a three-dimensional network from crossed or branched fibers and uncrossed spherical particles that filled the mesh space. The density of network, thickness of fibers, and properties of synthesized biomaterials depended on the polysaccharide type, charged degree of their macromolecule, and concentration. By varying these parameters, it was possible to manipulate the structural organization of hybrid polysaccharide-silica nanocomposites.

Hybrid, composite, and complex biomaterials
Prestwich, G. D. and H. Matthew (2002), Ann N Y Acad Sci 961: 106-8.

Hydraulic permeability of polyglycolic acid scaffolds as a function of biomaterial degradation
Li, J. and A. F. Mak (2005), J Biomater Appl 19(3): 253-66.
Abstract: Using a simple experimental setup, the hydraulic permeability of fibrous nonwoven polyglycolic acid (PGA) scaffolds is studied after different degradation durations in PBS. The hydraulic permeability of the scaffolds increased with the degradation time. After being incubated for about 4 weeks, the permeability of the scaffold begins to drop. It is noted that the PGA scaffold apparently begins to contract and cannot maintain its original shape after 4 weeks of degradation. These results underpin the understanding of the biotransport processes in the scaffolds during tissue engineering experiments.

Hydrogen peroxide for prevention of bacterial growth on polymer biomaterials
Alt, E., F. Leipold, et al. (1999), Ann Thorac Surg 68(6): 2123-8.
Abstract: BACKGROUND: Despite widespread use of potent antibiotics, infections of artificial implants and catheters are of increasing concern. We tested whether local treatment with 3% hydrogen peroxide (H2O2), long known as an inexpensive wound disinfectant, could prevent or reduce bacterial growth on polymer biomaterials. METHODS: Two-centimeter-long pieces of polyurethane and silicone tubing were contaminated with a standardized solution of Staphylococcus epidermidis (10(5)/mL) and then rinsed and wiped with saline (0.9%) solution. Bacterial growth was assessed after incubation at 37 degrees C for 24 hours. Bacterial colonies were compared for the following treatments: wiping only with saline; wiping with 1.5%, 2%, or 3% H2O2; pretreating biomaterials with 3% H2O2 and subsequent contamination for 2 and 4 hours without treatment after contamination; and contamination of tubings 1 month after pretreatment with 3% H2O2. The effect of 3% H2O2 was also assessed on contamination with Escherichia coli. RESULTS: Bacterial growth was reduced by more than 99% when the contaminated tubes were treated with 3% H2O2 compared with saline control (p < 0.001). Lower concentrations of H2O2 were less effective. The length of the contamination period had no influence on the effectiveness of H2O2 when used on polyurethane but did with silicone tubings. Pretreatment with H2O2 1 month before contamination still reduced bacterial growth rate by 90% on polyurethane and by 75% on silicone tubings. Comparable effects on bacterial growth rate were observed for staphylococci (-90%, p < 0.001) and escherichiae (-90%, p < 0.001). CONCLUSIONS: Local treatment with 3% H2O2 significantly reduced bacterial growth on polymer biomaterials even for 1 month after treatment. This finding might influence clinical strategies of prevention of foreign body infection.

Hydrogen peroxide mediates defence responses induced by chitosans of different molecular weights in rice
Lin, W., X. Hu, et al. (2005), J Plant Physiol 162(8): 937-44.
Abstract: To investigate the mechanisms whereby treatment with chitosan (CHN) is observed to increase the capacity of plants to resist pathogens, CHNs of different molecular weights (MWs) prepared by enzyme hydrolysis were used to treat rice cells in suspension culture and also rice seedlings. The results obtained with cultured cells showed that in this material CHN treatment could trigger a set of defence responses, including the production of hydrogen peroxide (H2O2), increases in the activities of phenylalanine ammonialyase (PAL; EC 4.3.1.5) and chitinase (CHI; EC 3.2.1.14), increases in transcription of defence-related genes beta-1,3-glucanase (glu) and chitinase (chi) and accumulation of pathogen-related protein (PR1). Furthermore, CHNs of different MWs were observed to have different capacities to induce defence responses. CHNs of low MWs were more effective at inducing the described defence responses than those of higher MWs. Enhanced defence against rice blast pathogen Magnaporthe grisea 97-23-2D1 was observed in rice seedlings treated with low MW CHNs compared to seedlings treated with higher MW CHNs. In all cases, suppressing the production of H2O2 by adding scavengers dimethylthiourea (DMTU), 2,5-dihydroxycinnamic acid methyl ester (DHC), catalase (Cat) or ascorbate (As) blocked the defence responses. These results indicate that CHNs of low MWs have a greater capacity to induce the production of H2O2, thus resulting in stronger defence responses, than those with higher MWs.

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