Biomaterials (v.27, #20)
Editorial board (CO2).
Biocompatibility of alginate–poly-l-lysine microcapsules for cell therapy by Gorka Orive; Susan K. Tam; José Luis Pedraz; Jean-Pierre Hallé (3691-3700).
Cell microencapsulation holds promise for the treatment of many diseases by the continuous delivery of therapeutic products. The biocompatibility of the microcapsules and their biomaterials components is a critical issue for the long-term efficacy of this technology. The objective of this paper is to provide detailed information about the principal factors affecting the biocompatibility of alginates and alginate–poly-l-lysine microcapsules, which are the most frequently employed biomaterials and encapsulation devices for cell immobilization, respectively. Some of these factors include the alginate composition and purification, the selection of the polycation, the interactions between the alginates and the polycation, the microcapsule fabrication process, the uniformity of the devices and the implantation procedure. Improved knowledge will lead to the production of standardized transplantation-grade biomaterials and biocompatible microcapsules.
Keywords: Alginate; Microencapsulation; Biocompatibility; Purification; Poly-l-lysine; Islets of Langerhans;
Processing and mechanical properties of HA/UHMWPE nanocomposites by Liming Fang; Yang Leng; Ping Gao (3701-3707).
A hydroxyapatite (HA) particulate reinforced ultrahigh molecular weight polyethylene (UHMWPE) nanocomposite was fabricated by compounding HA and UHMWPE mixtures in paraffin oil using twin-screw extrusion and then compression molding. Scanning electron microscope images revealed that HA aggregates were broken down to nano-sized particles and homogeneously dispersed in UHMWPE by the combined processes of twin-screw extrusion and UHMWPE swelling treatment. Transmission electron microscope images indicated the HA particles and UHMWPE matrix were intimately contacted through mechanical interlocking. The composite with the HA volume fraction of 0.23 exhibited a Young's modulus nine times higher than that of UHMWPE, while the composite maintained the excellent toughness feature of UHMWPE. The fracture strain reached over 300%, significantly higher than other types of biocomposites.
Keywords: Nanocomposite; Polyethylene; Hydroxyapatite; Mechanical properties; Extrusion;
The effect of surface area on the degradation rate of nano-fibrous poly(l-lactic acid) foams by Victor J. Chen; Peter X. Ma (3708-3715).
In vitro hydrolytic degradation behavior was examined for nano-fibrous (NF) poly(l-lactic acid) (PLLA) foams prepared by phase separation. NF foams were incubated in phosphate-buffered saline at 37 °C for 15 months. Upon removal, changes in mass, molar mass, morphology, BET specific surface area, mechanical properties, and thermal properties were compared with those of similarly incubated solid-walled (SW) PLLA foams. Initial surface area in NF foams was over 80 times higher than in SW foams. During incubation, NF surface area decreased steadily, only possessing 17% of the original specific surface area after 15 months, SW surface area stayed constant throughout. While molar mass decreased for both types of samples, degradation was much more rapid in NF foams. In NF foams, overall mass loss was 51% while mass loss in SW foams was only 6% after 15 months. Morphology of NF foams began as a mesh of fibers, and became increasingly porous as fibers began to aggregate, thus diminishing the mechanical properties. In SW foams, morphology was non-fibrous and remained unchanged which helped maintain their mechanical properties. These results suggest that the high surface area in NF foams accelerated the rate of hydrolytic degradation.
Keywords: Nano; Fiber; Polylactic acid; Degradation; Autocatalysis; Hydrolysis;
Integrin β 1 silencing in osteoblasts alters substrate-dependent responses to 1,25-dihydroxy vitamin D3 by Liping Wang; Ge Zhao; Rene Olivares-Navarrete; Bryan F. Bell; Marco Wieland; David L. Cochran; Zvi Schwartz; Barbara D. Boyan (3716-3725).
Surface microroughness increases osteoblast differentiation and enhances responses of osteoblasts to 1,25-dihydroxyvitamin D3 [1α,25(OH)2D3]. The observations that β 1 integrin expression is increased in osteoblasts grown on Ti substrates with rough microarchitecture, and that it is regulated by 1α,25(OH)2D3 in a surface-dependent manner, suggest that β 1 may play a role in mediating osteoblast response. To test this hypothesis, we silenced β 1 expression in MG63 human osteoblast-like cells using small interfering RNA (siRNA) and examined the responses of the β 1-silenced osteoblasts to surface microtopography and 1α,25(OH)2D3. To better understand the role of β 1, MG63 cells were also treated with two different monoclonal antibodies to human β 1 to block ligand binding. β 1-silenced MG63 cells grown on a tissue culture plastic had reduced alkaline phosphatase activity and levels of osteocalcin, transforming growth factor β1, prostaglandin E2, and osteoprotegerin in comparison with control cells. Moreover, β 1-silencing inhibited the effects of surface roughness on these parameters and partially inhibited effects of 1α,25(OH)2D3. Anti β 1 antibody AIIB2 had no significant effect on cell number and osteocalcin, but decreased alkaline phosphatase; MAB2253Z caused dose-dependent decreases in cell number and alkaline phosphatase and an increase in osteocalcin. Effects of 1α,25(OH)2D3 on cell number and alkaline phosphatase were reduced and effects on osteocalcin were increased. These findings indicate that β 1 plays a major and complex role in osteoblastic differentiation modulated by either surface microarchitecture or 1α,25(OH)2D3. The results also show that β 1 mediates, in part, the synergistic effects of surface roughness and 1α,25(OH)2D3.
Keywords: Titanium; Integrin; Microstructure; Osteoblast; siRNA;
Bias-assisted in vitro calcification of calcium disilicide growth layers on spark-processed silicon by Vladimir V. Seregin; Jeffery L. Coffer (3726-3737).
A dry-etch spark ablation method was used to produce calcium disilicide (CaSi2/Si) layers on silicon (Si) surfaces for the electrochemical growth of apatitic phosphates (calcium phosphate, CaP). CaSi2/Si composite electrodes readily calcify in vitro under the application of a small electric potential, and with proper treatment, the electrodeposition of CaP is localized to the sparked areas. In addition to increasing the local concentration of calcium, interfacial layers of CaSi2 on Si exhibit exceptional site selectivity towards CaP formation under bias due to the difference in conductivity between Si and CaSi2. The proposed mechanism for bias-assisted biomineralization of CaSi2/Si layers on spark-processed Si accounts for the physicochemical properties of deposited CaP films. This work also describes routes to surface modification of calcified composite electrodes with medicinally relevant compounds such as alendronate and norfloxacin. To assess the suitability of this material as a drug-delivery platform, release of the latter compound was also monitored as a function of time.
Keywords: Calcium disilicide; Spark ablation; Bioelectronics; Calcification; Drug delivery;
The role of cell signaling defects on the proliferation of osteoblasts on the calcium phosphate apatite thin film by Yun-Jung Lee; Jea Seung Ko; Hyun-Man Kim (3738-3744).
The intracellular signal transduction controlling the proliferation of osteoblastic cells on a thin film of poorly crystalline calcium phosphate apatite crystals (PCA) was studied in vitro. The PCA thin film was prepared on polystyrene culture dishes or cover glasses using phosphate-buffered calcium ion solution, which was made oversaturated by heating an undersaturated solution prepared at low temperature. The PCA thin film was used for cell culture without additional surface treatment. Several differences were found between the cells plated on a cell culture dish and the cells cultured on the PCA surface. Entry into S-phase of the cell cycle was markedly delayed and there was a low proliferation rate of osteblast. On the PCA thin film, the cells spread in a more slender shape. Also, the formation of focal adhesions and stress fibers, examined using immunocytochemical staining, was strikingly weaker in the cells on PCA. The activation of focal adhesion kinase (FAK) was low as well. Expression of cyclins D1 and E was also lower. The Ras–Extracellular signal-regulated kinase (ERK)–MAP kinase signaling pathway was weakly activated by stimulation with serum. These results demonstrate that the low cell proliferation on the PCA surface appears to be due to insufficient activation of signaling that forces the cell cycle to progress and this may be due to weak adhesion signaling in cells on that surface.
Keywords: Calcium phosphate coating; Biocompatibility; Osteoblast; Bone tissue engineering;
Effect of surface-adsorbed proteins and phosphorylation inhibitor AG18 on intracellular protein expression in adherent macrophages by Sean T. Zuckerman; Weiyuan John Kao (3745-3757).
Macrophages are believed to play an important role in the host inflammatory response to implanted biomaterials. However, the mechanism of macrophage adhesion to protein-adsorbed substrates and the subsequent activation and inflammation is unresolved. Previously the effect of various surface-adsorbed proteins and increasing concentrations of phosphorylation inhibitor AG18 on intracellular protein expression levels in adherent human monocytic cell line U937 was identified using SDS-PAGE and densitometry. The protein ligands and AG18 concentrations up or down regulated the expression of a set of proteins ranging from ∼200 to ∼23 kDa. In the present work, HPLC coupled tandem mass spectroscopy (LC/MS) was used to identify proteins in these bands. We hypothesized that key proteins in macrophage adhesion and activation could be identified by observing protein expression resulting from various surface-adsorbed ligands and AG18 concentrations. Increasing concentrations of AG18 down or up regulate protein expression in adherent U937 on PBS-adsorbed TCPS at ∼52, ∼42 and ∼23 kDa. AG18 concentrations had no effect on cells on albumin (Alb)-adsorbed surfaces but regulated different protein expression in adherent U937 on fibronectin (FN)-adsorbed TCPS at 40 and 80 μm AG18. Both Alb and FN regulate distinct sets of proteins in adherent cells as surface-adsorbed ligands. Based on the data from LC/MS, both surface associated ligand and increasing concentrations of AG18 modulate shifts in intracellular signaling.
Keywords: LC/MS; U937; Proteomics; Tyrosine phosphorylation; Inflammation; Fibronectin;
The effect of d,l-β-hydroxybutyric acid on cell death and proliferation in L929 cells by Shan Cheng; Guo-Qiang Chen; Michael Leski; Bing Zou; Yang Wang; Qiong Wu (3758-3765).
As a prerequisite for tissue engineering applications, researchers must understand the effect on local cell types of the degradation products of biodegradable polymers. Polyhydroxybutyrate (PHB) has received special interest as an implant material, because it degrades to release a normal component of blood and tissue, D,L-β-hydroxybutyrate (HB). We report that HB (0.02 g/l) promoted cell proliferation in cultured L929 cells plated at high cell density (1×105 cells/well) but not lower cell densities. While HB did not affect cell cycle progression, it significantly inhibited cell death. HB treatment prevented necrosis, reducing cell membrane permeability 4 h following serum withdrawal from the medium, and for all subsequent time points. In summary, HB promotes proliferation of L929 cells in high-density cultures by preventing apoptotic and necrotic cell death. This property makes biodegradable polymers containing HB, such as PHBHHx, attractive candidates for tissue engineering applications, especially those requiring the regeneration of large numbers of cells.
Keywords: Polyhydroxybutyrate; PHB; d,l-β-Hydroxybutyric acid; Cell proliferation; Necrosis; Apoptosis;
Cluster analysis and gene expression profiles: A cDNA microarray system-based comparison between human dental pulp stem cells (hDPSCs) and human mesenchymal stem cells (hMSCs) for tissue engineering cell therapy by Yoichi Yamada; Atsushi Fujimoto; Akira Ito; Ryoko Yoshimi; Minoru Ueda (3766-3781).
We investigated gene expression patterns and functional classifications regarding the clusters of human dental pulp stem cells (hDPSCs) and human mesenchymal stem cells (hMSCs)—which possess a multipotent ability—because little is known about the precise moleculobiological clues by which these cells activate their differentiating ability or functionality to eventually form dentin and bone, respectively. We first verified the expressions of the alkaline phosphatase (ALP) gene, dentin matrix protein 1 (DMP-1), and dentinsialophosphoprotein (DSPP) by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and consequently discovered the high expressions of these genes. Total RNA was also followed by hybridization with a human microarray system consisting of 12,814 genes. Analyses of gene expression patterns indicated several genes which encode extracellular matrix components, cell adhesion molecules, growth factors, and transcription regulators. Functional and clustering analyses of differences in gene expression levels revealed cell signaling, cell communication, or cell metabolism. In the future, information on the gene expression patterns of hDPSCs and hMSCs might be useful in determining the detailed functional roles of the relevant genes and applicable to stem cell therapies, and these cells could also be used as multipotent cell sources for gene technology and tissue engineering technology.
Keywords: Human dental pulp stem cells (hDPSCS); Human mesenchymal stem cells (hMSCS); Cluster analysis; Gene expression; Tissue engineering;
Electrospun three-dimensional hyaluronic acid nanofibrous scaffolds by Yuan Ji; Kaustabh Ghosh; Xiao Zheng Shu; Bingquan Li; Jonathan C. Sokolov; Glenn D. Prestwich; Richard A.F. Clark; Miriam H. Rafailovich (3782-3792).
A three-dimensional (3D) hyaluronic acid (HA) nanofibrous scaffold was successfully fabricated to mimic the architecture of natural extracelluar matrix (ECM) based on electrospinning. Thiolated HA derivative, 3,3′-dithiobis(propanoic dihydrazide)-modified HA (HA-DTPH), was synthesized and electrospun to form 3D nanofibrous scaffolds. In order to facilitate the fiber formation during electrospinning, Poly (ethylene oxide) (PEO) was added into the aqueous solution of HA-DTPH at an optimal weight ratio of 1:1. The electrospun HA-DTPH/PEO blend scaffold was subsequently cross-linked through poly (ethylene glycol)-diacrylate (PEGDA) mediated conjugate addition. PEO was then extracted in DI water to obtain an electrospun HA-DTPH nanofibrous scaffold. NIH 3T3 fibroblasts were seeded on fibronectin-adsorbed HA-DTPH nanofibrous scaffolds for 24 h in vitro. Fluorescence microscopy and laser scanning confocal microscopy revealed that the 3T3 fibroblasts attached to the scaffold and spread, demonstrating an extended dendritic morphology within the scaffold, which suggests potential applications of HA-DTPH nanofibrous scaffolds in cell encapsulation and tissue regeneration.
Keywords: Hyaluronic acid; Electrospinning; Nanofibers; Hydrogel; Tissue engineering;
Mechanical properties and in vitro biocompatibility of porous zein scaffolds by Shengju Gong; Huajie Wang; Qingshen Sun; Song-Tao Xue; Jin-Ye Wang (3793-3799).
A porous scaffold utilizing hydrophobic protein zein was prepared by the salt-leaching method for tissue engineering. The scaffolds possessed a total porosity of 75.3–79.0%, compressive Young's modulus of (28.2±6.7)MPa–(86.6±19.9)MPa and compressive strength of (2.5±1.2)MPa–(11.8±1.7)MPa, the percentage degradation of 36% using collagenase and 89% using pepsin during 14 days incubation in vitro. The morphology of pores located on the surface and within the porous scaffolds showed good pore interconnectivity by scanning electron microscopy (SEM). Rat mesebchymal stem cells (MSCs) could adhere, grow, proliferate and differentiate toward osteoblasts on porous zein scaffold. With the action of dexamethasone, the cells showed a relative higher activity of alkaline phosphatase (ALP) and a higher proliferating activity ( p < 0.05 ) than those of MSCs without dexamethasone.
Keywords: Scaffold; Zein; Mechanical properties; Porosity; Degradation; MSCs;
Fabrication and characterization of permeable degradable poly(dl-lactide-co-glycolide) (PLGA) hollow fiber phase inversion membranes for use as nerve tract guidance channels by Xuejun Wen; Patrick A. Tresco (3800-3809).
Biodegradable permeable poly(dl-lactide-co-glycolide) (PLGA) hollow fiber membranes (HFMs) were fabricated using a wet phase inversion technique. By varying several parameters, such as the spinneret size, solvent and non-solvent pair, polymer concentration, flow rate, precipitation method, drop height, and small molecular pore-forming agents, PLGA HFMs with variable sizes, surface morphologies, porosities, and diffusive permeability were obtained. Under simulated physiological conditions in vitro, PLGA HFMs exhibited a degradation profile to accommodate nervous system regeneration and axonal outgrowth. While accelerated degradation resulted in substantial molecular weight loss starting at 2 weeks and loss of selective permeability at 3 weeks, PLGA HFMs maintained gross structural integrity in the first 4 weeks, followed by sharp weight loss at 6 weeks and complete disappearance at about 8 weeks. When compared to the raw PLGA material in a pellet form, which underwent heterogeneous degradation, the PLGA HFMs exhibited a homogeneous degradation where the surface and bulk degraded at approximately the same rate, and an overall lower degradation rate. Our results indicate that using a wet phase inversion technique, degradable HFMs with variable size, inner and outer surface morphologies, porosity, and permeability with potential applications for nerve tract guidance conduits can be fabricated.
Keywords: Biodegradation; Hollow fiber membrane; Nerve guidance; Degradable polyester; Permeability;
The effect of a fibrin-fibronectin/β-tricalcium phosphate/recombinant human bone morphogenetic protein-2 system on bone formation in rat calvarial defects by Sung-Jae Hong; Chang-Sung Kim; Dong-Kwan Han; Ik-Hyun Cho; Ui-Won Jung; Seong-Ho Choi; Chong-Kwan Kim; Kyoo-Sung Cho (3810-3816).
In spite of good prospects for bone morphogenetic proteins (BMP) applications, an ideal carrier system for BMPs has not yet been identified. The purpose of this study was to evaluate the osteogenic effect of a fibrin-fibronectin sealing system (FFSS) combined with β-tricalcium phosphate (β-TCP) as a carrier system for recombinant human bone morphogenetic proteins (rhBMP-2) in the rat calvarial defect model.Eight-millimeter critical-size calvarial defects were created in 100 male Sprague–Dawley rats. The animals were divided into five groups of 20 animals each. The defects were treated with rhBMP-2/FFSS, rhBMP-2/FFSS/β-TCP, FFSS and FFSS/β-TCP carrier control or were left untreated as a sham-surgery control. Defects were evaluated by histologic and histometric parameters following a 2- and 8-week healing interval (10 animals/group/healing intervals).The FFSS/β-TCP carrier group was significantly greater in new bone area at 2 weeks ( p < 0.05 ) and new tissue area at 2 and 8 weeks ( p < 0.01 ) relative to the FFSS carrier group. New bone and new tissue area in the rhBMP-2/FFSS/β-TCP group were significantly greater than in the rhBMP-2/FFSS group at 8 weeks ( p < 0.01 ). On histologic observation, FFSS remnants were observed at 2 weeks, but by 8 weeks, the FFSS appeared to be completely resorbed. rhBMP-2 combined with FFSS/β-TCP produced significantly more new bone and new tissue formation in this calvarial defect model. In conclusion, FFSS/β-TCP may be considered as an available carrier for rhBMP-2.
Keywords: Bone regeneration; Carrier; Bone morphogenetic protein-2; Carrier; Fibrin-fibronectin sealing system; β-tricalcium phosphate; Rat calvarial defect model;
Combinatorial screen of the effect of surface energy on fibronectin-mediated osteoblast adhesion, spreading and proliferation by Scott B. Kennedy; Newell R. Washburn; Carl George Simon; Eric J. Amis (3817-3824).
In order to accelerate tissue-engineering research, a combinatorial approach for investigating the effect of surface energy on cell response has been developed. Surface energy is a fundamental material property that can influence cell behavior. Gradients in surface energy were created by using an automated stage to decelerate a glass slide coated with a self-assembled monolayer (SAM, n-octyldimethylchlorosilane) beneath a UV lamp such that the SAM is exposed to the UV-light in a graded fashion. UV exposure causes oxidation of the SAM such that a longer exposure correlates with increased hydrophilicity. This approach yielded substrates having a linear gradient in surface energy ranging from 23 to 62 mN/m (water contact angles ranging from 25° to 95°). Using the gradient specimen approach enables all surface energies from 23 to 60 mN/m to be screened on each slide. Before cell culture, surface energy gradients were coated with fibronectin to allow a study of the effect of surface energy on fibronectin-mediated cell response. Cells were seeded on the fibronectin-coated gradients and adhesion, spreading and proliferation were assessed with automated fluorescence microscopy. Surface energy did not affect initial cell adhesion at 8 h. However, the rate of proliferation was linearly dependent on surface energy and increased with increasing hydrophobicity. Cell spread area was unaffected by changes in surface energy over the majority of the gradient although cells were significantly smaller on the most hydrophilic region. These results show that fibronectin-mediated cell spreading and proliferation are dependent on surface energy and establish a new combinatorial approach for screening cell response to changes in surface energy.
Keywords: Cell adhesion; Cell proliferation; Cell spreading; Osteoblast; Self-assembly; Surface energy;
Effects of hyaluronan and SPARC on fibroproliferative events assessed in an in vitro bladder acellular matrix model by Allison L. Brown; Murice J. Ringuette; Glenn D. Prestwich; Darius J. Bagli; Kimberly A. Woodhouse (3825-3835).
Bladder acellular matrix (BAM) is a promising candidate for urinary biomaterials development. In the current work we have modified the BAM construct to include two biologically active components; hyaluronan (HA) and a peptide (SP4.2) derived from secreted protein, acidic, rich in cysteine (SPARC), a matricellular glycoprotein. In order to assess the potential of an HA/SP4.2 modified BAM to influence cellular functions associated with bladder healing, experiments were conducted to evaluate the individual and combined effects of these molecules on in vitro fibroproliferative endpoints within a co-culture model.Thiol-modified HA (246 kDa, 15 mg/ml)±SP4.2 (200 μm) was incorporated and cross-linked into BAM disks through disulfide bond formation. The following scaffolds compositions were then evaluated in a bladder smooth muscle cell (SMC)–urothelial (UEC) cell co-culture model: BAM unmodified; BAM+HA, BAM+SP4.2 (media addition); BAM+HA+SP4.2 (media addition); BAM+HA+SP4.2 (matrix incorporated). At 3, 7 and 14 days post-seeding, SMC-mediated matrix contraction and gelatinolytic activity were evaluated.HA-modified BAM exhibited a significantly higher degree of contraction and gelatinase activity compared to unmodified BAM. In contrast, addition of SP4.2 to BAM produced a negligible effect on contraction, while significantly reducing gelatinase activity. Matrices containing both molecules displayed significant increases in contraction, while gelatinase activity was dependent upon the method of peptide delivery.These results demonstrate that both HA and SP4.2 have significant, yet distinct effects on the contractile and proteolytic activity of bladder SMCs and suggest that a modified BAM may be capable of modulating processes associated with post-surgical graft contracture and scar formation.
Keywords: Bladder tissue engineering; Co-culture; Hyaluronan/hyaluronic acid; Extracellular matrix; Matrix metalloproteinase; Smooth muscle cell;
Standardization criteria for an ischemic surgical model of acute hepatic failure in pigs by Martin Nieuwoudt; Richarda Kunnike; Mario Smuts; Johan Becker; G. Frik Stegmann; Christelle Van der Walt; Johan Neser; Schalk Van der Merwe (3836-3845).
The establishment of a reliable large animal model of acute hepatic failure (AHF) is critical for the evaluation of supportive therapies such as bioartificial liver support systems (BALSS). Large animal surgical models bear some resemblance to the clinical syndrome of AHF in humans. However, these are inherently complex and are predisposed to inter-individual variation. The development of considerable skill is required and no precise standardization criteria for such models have been defined as yet. This study investigates a surgically induced ischemic model of AHF in 15 female Landrace pigs. A large set of systemic and biochemical variables were measured. The absolute values of systemic variables during the surgery and the rates of change of the biochemical variables following the surgery were correlated with the duration of survival of each animal. A multivariate prognosis was revealed, with several variables simultaneously determining survival. Those of importance in the surgical period included the duration of portal occlusion, the pulse rate during this time, the total blood lost, the mean arterial pressure and the body temperature after the procedure. In the post-surgical period the rates of change of blood ammonia, branch chain amino acids, haemoglobin, hematocrit, body temperature, total urinary excretion and mean blood pH all demonstrated some importance to survival. Based on the above and clinical experience, standardization criteria specific for this model were defined. Additional variables that posses value in AHF but demonstrate lower correlations with survival were also included, e.g. the prothrombin time, clotting factors, liver enzymes, potassium, bilirubin, creatinine and lactate. Valuable procedural information was generated, which stabilized the model, limited unnecessary blood sampling and cut laboratory costs. The criteria allow the early exclusion of compromized animals and the identification of those demonstrating characteristics predictive of longer survival prior to BALSS connection. This is likely to allow more accurate comparisons between small treatment and control groups. In effect, the above are the converse of the King's college criteria (for humans) as they are designed to exclude animals with prognoses that are insufficiently bleak or due to factors other than AHF. The statistical analysis employed also identified the variables that in future may allow the evaluation of prognosis in real-time.
Keywords: Criteria; Ischemic surgical model; Acute liver failure; Pig;
Novel delivery system based on complexation hydrogels as delivery vehicles for insulin–transferrin conjugates by Nikhil J. Kavimandan; Elena Losi; Nicholas A. Peppas (3846-3854).
A variety of approaches have been investigated to address the problems associated with oral insulin delivery, but the bioavailability of oral insulin is still low. Insulin is rapidly degraded by the enzymes in the GI tract and is not transported across the epithelial barrier easily. The oral insulin formulation developed in this work makes use of complexation hydrogels for oral delivery of insulin bioconjugates. The insulin bioconjugates synthesized in this work consist of insulin bound to transferrin molecule which can be uptaken by the epithelial cells. The conjugates can increase the permeability of insulin across the epithelial barrier by receptor-mediated transcytosis. The transferrin in the conjugate is also shown to stabilize insulin in the presence of intestinal enzymes. Use of complexation hydrogels for delivery of insulin–transferrin conjugate may greatly increase the bioavailability of oral insulin. This is because, the complexation hydrogels are known to exhibit characteristics that make them ideal candidates for oral protein delivery. They can also inhibit the degradation of insulin in the GI tract. Thus, combination of these two approaches may provide an innovative platform for oral insulin delivery.
Keywords: Insulin; Transferrin; Protein delivery; Hydrogels;
Biotechnological traps for the reduction of inflammation due to cardiopulmonary bypass operations by Valentina Grano; Franca Salamino; Edon Melloni; Roberto Minafra; Eliana Regola; Nadia Diano; Carla Nicolucci; Angelina Attanasio; Gianantonio Nappi; Maurizio Cotrufo; Lucio Maresca; Natale Gaspare De Santo; Damiano Gustavo Mita (3855-3862).
Cardiopulmonary bypass induces a systemic inflammatory response (SIR), characterized by the activation of cellular and humoral elements, with concomitant release of neutrophil elastase and matrix-metallo proteinases.In the present study, the protease release during extracorporeal circulation in 28 patients undergoing cardiac surgical operations was monitored using casein zymography. A peak in protease activity was found in all patients at the end of cardiopulmonary bypass. Plasma samples of patients were allowed to interact with different traps obtained by immobilizing different protease inhibitors on specific carriers. α 1-Antitpypsin, Bovine Pancreatic Trypsin Inhibitor, Elastatinal or Leupeptin were used as inhibitors and were covalently immobilized by diazotization or by condensation. A reduction in the proteolytic activity of the plasma samples was observed after interaction with the different traps. The most efficient traps, i.e. the ones displaying greatest power to inhibit protease activity, were those obtained by immobilizing Bovine Pancreatic Trypsin Inhibitor and Leupeptin. The biocompatibility of traps was also tested.Results show that protease activity in blood can be decreased by our protease traps.
Keywords: Extracorporeal circulation; Blood flow; Surface grafting; Inflammation; Cell activation;
Effect of RGD secondary structure and the synergy site PHSRN on cell adhesion, spreading and specific integrin engagement by Sarah E. Ochsenhirt; Efrosini Kokkoli; James B. McCarthy; Matthew Tirrell (3863-3874).
The relationship between the form of cell adhesion, ligand presentation, and cell receptor function was characterized using model Langmuir–Blodgett supported films, containing lipid-conjugated peptide ligands, in which isolated variables of the ligand presentation were systematically altered. First, the conformation of an adhesive Arginine–Glycine–Aspartic acid (RGD) peptide was varied by synthesizing linear and looped RGD peptide-containing amphiphiles and subsequently measuring the impact on the function of human umbilical vein endothelial cells. Secondly, the contribution of non-contiguous ligands to cellular engagement was assessed using multi-component biomimetic films. The peptide amphiphiles were composed of fibronectin-derived headgroups—GRGDSP, and its synergy site Pro–His–Ser–Arg–Asn (PHSRN)—attached to hydrocarbon tails. The peptide amphiphiles were diluted using polyethylene glycol (PEG) amphiphiles, where PEG inhibited non-specific cell adhesion. Cells adhered and spread on GRGDSP/PEG systems in a dose-dependent manner. The presentation of GRGDSP influenced integrin cell surface receptor specificity. Results demonstrated that β 1-containing integrins mediated adhesion to the linear GRGDSP presentation to a greater extent than did the α v β 3 integrin, and looped GRGDSP preferentially engaged α v β 3. GRGDSP/PHSRN/PEG mixtures that closely mimicked the RGD–PHSRN distance in fibronectin, enhanced cell spreading over their two-component analogues. This study demonstrated that controlling the microenvironment of the cell was essential for biomimetics to modulate specific binding and subsequent signaling events.
Keywords: Looped RGD; Cyclic RGD; PHSRN; HUVEC; α5β1 integrin; α v β 3 integrin;