Three-dimensional printing and decellularized-extracellular-matrix based methods for advances in artificial blood vessel fabrication: A review.

Blood vessels are the tubes through which blood flows and are divided into three types: millimeter-scale arteries, veins, and capillaries as well as micrometer-scale capillaries. Arteries and veins are the conduits that carry blood, while capillaries are where blood exchanges substances with tissues. Blood vessels are mainly composed of collagen fibers, elastic fibers, glycosaminoglycans and other macromolecular substances. There are about 19 feet of blood vessels per square inch of skin in the human body, which shows how important blood vessels are to the human body. Because cardiovascular disease and vascular trauma are common in the population, a great number of researches have been carried out in recent years by simulating the structures and functions of the person's own blood vessels to create different levels of tissue-engineered blood vessels that can replace damaged blood vessels in the human body. However, due to the lack of effective oxygen and nutrient delivery mechanisms, these tissue-engineered vessels have not been used clinically. Therefore, in order to achieve better vascularization of engineered vascular tissue, researchers have widely explored the design methods of vascular systems of various sizes. In the near future, these carefully designed and constructed tissue engineered blood vessels are expected to have practical clinical applications. Exploring how to form multi-scale vascular networks and improve their compatibility with the host vascular system will be very beneficial in achieving this goal. Among them, 3D printing has the advantages of high precision and design flexibility, and the decellularized matrix retains active ingredients such as collagen, elastin, and glycosaminoglycan, while removing the immunogenic substance DNA. In this review, technologies and advances in 3D printing and decellularization-based artificial blood vessel manufacturing methods are systematically discussed. Recent examples of vascular systems designed are introduced in details, the main problems and challenges in the clinical application of vascular tissue restriction are discussed and pointed out, and the future development trends in the field of tissue engineered blood vessels are also prospected.

Tangential flow filtration facilitated fractionation and PEGylation of low and high-molecular weight polymerized hemoglobins and their biophysical properties.

Various types of hemoglobin (Hb)-based oxygen carriers (HBOCs) have been developed as red blood cell substitutes for treating blood loss when blood is not available. Among those HBOCs, glutaraldehyde polymerized Hbs have attracted significant attention due to their facile synthetic route, and ability to expand the blood volume and deliver oxygen. Hemopure®, Oxyglobin®, and PolyHeme® are the most well-known commercially developed glutaraldehyde polymerized Hbs. Unfortunately, only Oxyglobin® was approved by the FDA for veterinary use in the United States, while Hemopure® and PolyHeme® failed phase III clinical trials due to their ability to extravasate from the blood volume into the tissue space which facilitated nitric oxide scavenging and tissue deposition of iron, which elicited vasoconstriction, hypertension and oxidative tissue injury. Fortunately, conjugation of poly (ethylene glycol) (PEG) on the surface of Hb is capable of reducing the vasoactivity of Hb by creating a hydration layer surrounding the Hb molecule, which increases its hydrodynamic diameter and reduces tissue extravasation. Several commercial PEGylated Hbs (MP4®, Sanguinate®, Euro-PEG-Hb) have been developed for clinical use with a longer circulatory half-life and improved safety compared to Hb. However, all of these commercial products exhibited relatively high oxygen affinity compared to Hb, which limited their clinical use. To dually address the limitations of prior generations of polymerized and PEGylated Hbs, this current study describes the PEGylation of polymerized bovine Hb (PEG-PolybHb) in both the tense (T) and relaxed (R) quaternary state via thiol-maleimide chemistry to produce an HBOC with low or high oxygen affinity. The biophysical properties of PEG-PolybHb were measured and compared with those of commercial polymerized and PEGylated HBOCs. T-state PEG-PolybHb possessed higher hydrodynamic volume and P50 than previous generations of commercial PEGylated Hbs. Both T- and R-state PEG-PolybHb exhibited significantly lower haptoglobin binding rates than the precursor PolybHb, indicating potentially reduced clearance by CD163 + monocytes and macrophages. Thus, T-state PEG-PolybHb is expected to function as a promising HBOC due to its low oxygen affinity and enhanced stealth properties afforded by the PEG hydration shell.

Function of Hemoglobin-Based Oxygen Carriers: Determination of Methemoglobin Content by Spectral Extinction Measurements.

Suspensions of hemoglobin microparticles (HbMPs) are promising tools as oxygen therapeutics. For the approval of clinical studies extensive characterization of these HbMPs with a size of about 750 nm is required regarding physical properties, function, pharmaco-kinetics and toxicology. The standard absorbance measurements in blood gas analyzers require dissolution of red blood cells which does not work for HbMP. Therefore, we have developed a robust and rapid optical method for the quality and functionality control of HbMPs. It allows simultaneous determination of the portion of the two states of hemoglobin oxygenated hemoglobin (oxyHb) and deoxygenated hemoglobin (deoxyHb) as well as the content of methemoglobin (metHb). Based on the measurement of collimated transmission spectra between 300 nm and 800 nm, the average extinction cross section of HbMPs is derived. A numerical method is applied to determine the composition of the HbMPs based on their wavelength-dependent refractive index (RI), which is a superposition of the three different states of Hb. Thus, light-scattering properties, including extinction cross sections can be simulated for different compositions and sizes. By comparison to measured spectra, the relative concentrations of oxyHb, deoxyHb, metHb are accessible. For validation of the optically determined composition of the HbMPs, we used X-ray fluorescence spectrometry for the ratio of Fe(II) (oxyHb/deoxyHb) and Fe(III) (metHb). High accuracy density measurements served to access heme-free proteins, size was determined by dynamic light scattering and analytical centrifugation and the shape of the HbMPs was visualized by electron and atomic force microscopy.

Wireless, battery-free optoelectronic systems as subdermal implants for local tissue oximetry.

Monitoring regional tissue oxygenation in animal models and potentially in human subjects can yield insights into the underlying mechanisms of local O2-mediated physiological processes and provide diagnostic and therapeutic guidance for relevant disease states. Existing technologies for tissue oxygenation assessments involve some combination of disadvantages in requirements for physical tethers, anesthetics, and special apparatus, often with confounding effects on the natural behaviors of test subjects. This work introduces an entirely wireless and fully implantable platform incorporating (i) microscale optoelectronics for continuous sensing of local hemoglobin dynamics and (ii) advanced designs in continuous, wireless power delivery and data output for tether-free operation. These features support in vivo, highly localized tissue oximetry at sites of interest, including deep brain regions of mice, on untethered, awake animal models. The results create many opportunities for studying various O2-mediated processes in naturally behaving subjects, with implications in biomedical research and clinical practice.

Fatty Acid Saturation of Albumin Used in Resuscitation Fluids Modulates Cell Damage in Shock: in vitro Results Using a Novel Technique to Measure Fatty Acid Binding Capacity.

The use of albumin for resuscitation has not proven as beneficial in human trials as expected from numerous animal studies. One explanation could be the practice of adding fatty acid (FA) during manufacture of pharmaceutical albumin. During ischemia, unbound free FAs (FFA) in the circulation could potentially induce cellular damage. We hypothesized that albumins with higher available binding capacities (ABC) for FFAs may prevent that damage. Therefore, we developed a technique to measure ABC, determined if pharmaceutical human serum albumin (HSA) has decreased ABC compared with FA-free bovine serum albumin (BSA), and if binding capacity would affect hemolysis when blood is mixed with exogenous FFA at levels similar to those observed in shock. The new assay used exogenous oleic acid (OA), glass fiber filtration, and a FFA assay kit. RBC hemolysis was determined by mixing 0 to 5 mM OA with PBS, HSA, FA-free BSA, or FA-saturated BSA and measuring plasma hemoglobin after incubation with human blood. 5% HSA contained 4.7±0.2 mM FFA, leaving an ABC of 5.0 ±â€Š0.6 mM, compared with FA-free BSAs ABC of 7.0 ±â€Š1.3 mM (P < 0.024). Hemolysis after OA was reduced with FA-free BSA but increased with FA-saturated BSA. HSA provided intermediate results. 25% solutions of FA-free BSA and HSA were more protective, while 25% FA-saturated BSA was more damaging than 5% solutions. These findings suggest that increased FA saturation may reverse albumin's potential benefit to lessen cellular damage and may explain, at least in part, its failure in human trauma studies.

Application of HBOCs electrophoretic method to detect a new blood substitute derived from the giant extracellular haemoglobin of lugworm.

Manipulation of blood and blood components is prohibited in sports by the World Anti-Doping Agency (WADA). This includes the use of blood substitutes to increase oxygen transport, like haemoglobin-based oxygen carriers (HBOCs), which are compounds derived from haemoglobin. Despite their medical interest, the first generation of HBOCs had serious adverse effects and was abandoned. However, new studies are now exploiting the properties of marine worm haemoglobins, which circulate as giant extracellular complexes with high oxygen-binding capacities. HEMOXYCarrier® (HC), developed by Hemarina, is one of the most advanced and promising HBOCs, and HC may become a tempting doping tool for athletes in the future. Here, HC detection in plasma/serum was evaluated with the method used to detect the first HBOCs, based on electrophoresis and heme peroxidase properties. An HC-derived product was identified in human plasma up to 72 h after in vitro incubation at 37 °C. HC degradation also induced methemalbumin formation. After injecting HC at the effective dose of 200 mg/kg into mice, the HC-derived product was detected only for a few hours and no accumulation of methemalbumin was observed. Due to this limited detection window in vivo, measuring specific worm globin degradation products by mass spectrometry might be an alternative for future anti-doping analyses. Copyright © 2016 John Wiley & Sons, Ltd.

Liposomes as potential masking agents in sport doping. Part 2: Detection of liposome-entrapped haemoglobin by flow cytofluorimetry.

This work presents an analytical procedure for the identification and characterization of liposome-entrapped haemoglobins, based on flow cytofluorimetry. Flow cytofluorimetric detection is carried out following labelling by two distinct fluorescent reagents, an anti-haemoglobin antibody, fluorescein isothiocyanate conjugated, and an anti-poly(ethylene glycol) antibody, streptavidin-phycoerythrin conjugated. This experimental strategy allows the detection of liposome-entrapped haemoglobins in aqueous media, including plasma; the efficacy of the proposed approach has been verified on whole blood samples added with the liposomal formulation (ex-vivo). Additionally, the proposed technique allows the characterization of several key parameters in the study of liposomal haemoglobins, including, for instance (1) the determination of the degree of haemoglobin entrapment by liposomes; (2) the poly(ethylene glycol) insertion efficiency; and (3) the evaluation of liposome-entrapped haemoglobins stability following storage at 4 °C, allowing to follow both the process of haemoglobin loss from liposomes and the liposome degradation. The procedure is proposed for the detection and characterization of liposome-entrapped haemoglobin formulations to control their misuse in sport, but is also suggested for further applications in biological and clinical laboratory investigations. Copyright © 2016 John Wiley & Sons, Ltd.

Amperometric homogeneous competitive immunoassay in a perfluorocarbon emulsion oxygen therapeutic (PEOT).

The effect of a perfluorocarbon emulsion oxygen therapeutic (PEOT) on the detection of the drugs theophylline and phenytoin was explored using a commercial enzyme multiplied immunoassay technique (EMIT®). The EMIT technique is based on the enzymatic production of NADH, which is typically detected in serum samples spectrophotometrically. Here, amperometry using the rotating disk electrode on a single drop of solution is demonstrated to detect theophylline and phenytoin in the presence of PEOT. In the study, 2,6-dichloroindophenol (DCIP) added to the immunoassay mixture is reduced by the NADH to DCIPH2. Oxidation of DCIPH2 is monitored electrochemically at +200 mV using a glassy carbon rotating disk electrode. Slopes of amperograms are proportional to the concentration of drug in the immunoassay sample. This technique yields excellent quantitative data in the therapeutic range for both drugs in 2-20% PEOT.

Low modulus biomimetic microgel particles with high loading of hemoglobin.

We synthesized extremely deformable red blood cell-like microgel particles and loaded them with bovine hemoglobin (Hb) to potentiate oxygen transport. With similar shape and size as red blood cells (RBCs), the particles were fabricated using the PRINT (particle replication in nonwetting templates) technique. Low cross-linking of the hydrogel resulted in very low mesh density for these particles, allowing passive diffusion of hemoglobin throughout the particles. Hb was secured in the particles through covalent conjugation of the lysine groups of Hb to carboxyl groups in the particles via EDC/NHS coupling. Confocal microscopy of particles bound to fluorescent dye-labeled Hb confirmed the uniform distribution of Hb throughout the particle interior, as opposed to the surface conjugation only. High loading ratios, up to 5 times the amount of Hb to polymer by weight, were obtained without a significant effect on particle stability and shape, though particle diameter decreased slightly with Hb conjugation. Analysis of the protein by circular dichroism (CD) spectroscopy showed that the secondary structure of Hb was unperturbed by conjugation to the particles. Methemoglobin in the particles could be maintained at a low level and the loaded Hb could still bind oxygen, as studied by UV-vis spectroscopy. Hb-loaded particles with moderate loading ratios demonstrated excellent deformability in microfluidic devices, easily deforming to pass through restricted pores half as wide as the diameter of the particles. The suspension of concentrated particles with a Hb concentration of 5.2 g/dL showed comparable viscosity to that of mouse blood, and the particles remained intact even after being sheared at a constant high rate (1000 1/s) for 10 min. Armed with the ability to control size, shape, deformability, and loading of Hb into RBC mimics, we will discuss the implications for artificial blood.

Hypervolemic infusion of Lumbricus terrestris erythrocruorin purified by tangential-flow filtration.

BACKGROUND: The hemoglobin of the earthworm Lumbricus terrestris (also known as erythrocruorin, or LtEc) is a naturally occurring high-molecular-weight protein assembly (3.6 MDa) that is extremely stable, resistant to oxidation, and transports oxygen similarly to human whole blood. Therefore, LtEc may serve as an alternative to donated human red blood cells. However, a suitable purification process must be developed to produce highly pure LtEc on a large scale that can be evaluated in an animal model to determine the safety and efficacy of LtEc. STUDY DESIGN AND METHODS: We used tangential-flow filtration (TFF), an easily scalable and affordable technique, to produce highly pure LtEc from earthworms. The purity, yield, methemoglobin level, viscosity, colloid osmotic pressure, O(2) binding equilibria, and ligand-binding kinetics of the purified LtEc were measured in vitro. The purified LtEc product was then evaluated in hamsters using a hypervolemic infusion model to establish its basic biocompatibility and detect any changes in microcirculatory and systemic variables. RESULTS: TFF was able to produce LtEc with high purity and yield (5-10 g/1000 worms). The purified LtEc product did not elicit hypertension or vasoconstriction when infused into hamsters. CONCLUSION: LtEc may be easily purified and safely transfused into hamsters in small amounts (0.5-1.5 g/dL final concentration in blood) without any noticeable side effects. Therefore, LtEc may serve as a very promising oxygen carrier for use in transfusion medicine.

Practical considerations in the development of hemoglobin-based oxygen therapeutics.

The development of hemoglobin based oxygen therapeutics (HBOCs) requires consideration of a number of factors. While the enabling technology derives from fundamental research on protein biochemistry and biological interactions, translation of these research insights into usable medical therapeutics demands the application of considerable technical expertise and consideration and reconciliation of a myriad of manufacturing, medical, and regulatory requirements. The HBOC development challenge is further exacerbated by the extremely high intravenous doses required for many of the indications contemplated for these products, which in turn implies an extremely high level of purity is required. This communication discusses several of the important product configuration and developmental considerations that impact the translation of fundamental research discoveries on HBOCs into usable medical therapeutics.

Preparation and function of poly(acrylic acid)s modified by supramolecular complex composed of porphinatoiron and a cyclodextrin dimer that bind diatomic molecules (O2 and CO) in aqueous solution.

Poly(acrylic acid) (PAA) is modified by 5-(4-ß-alanylaminophenyl)-10,15,20-tris(4-sulfonatophenyl) porphinatoiron(III) to yield iron porphyrin-bearing PAAs (FeP(n)s) through a condensation reaction. FeP(n)s were further functionalized by Py3CD, which is a per-O-methylated ß-cyclodextrin (CD) dimer with a pyridine linker and includes the porphyrin pendants to form ferric hemoCD-P(n)s. Ferrous hemoCD-P(3), having three porphyrin chromophores in a polymer chain, is shown to bind molecular oxygen (P(1/2)=7.9±1.4 Torr) in aqueous solution at pH 7.0 and 25 °C, affording oxy-hemoCD-P(3). Oxy-hemoCD-P(3) is biphasically autoxidized to ferric hemoCD-P(3), with 27% of the dioxygen adducts being rapidly oxidized. The rate of autoxidation of oxy-hemoCD-P(15), having 15 porphyrin chromophores in a polymer chain, was much faster than that of oxy-hemoCD-P(3), thus suggesting self-catalyzed autoxidation of oxy-hemoCD-P(n)s. Oxy-hemoCD-P(n)s are markedly stabilized by catalase, thereby indicating that hydrogen peroxide generated from oxy-hemoCD-P(n) accelerates the autoxidation. Most of the hemoCD-P(3) molecules injected into the femoral vein of a rat remained in the body, though about 16% of the hemoCD-P(3) molecules were excreted in the urine as a carbon monoxide adduct.

Assessment of total haemoglobin mass: can it detect erythropoietin-induced blood manipulations?

The purpose of the study was to reveal erythropoietin (epo) doping. It was recently suggested that the assessment of total haemoglobin mass (tHb) by means of the carbon monoxide re-breathing technique should be implemented in anti-doping work. Since epo may increase the haemoglobin concentration [Hb] simply by reducing plasma volume we injected eight human subjects with epo for 15 weeks and directly tested the feasibility hereof. Epo treatment increased [Hb] in all subjects at all time points (range 3.8-18.8%). In approximately half the subjects this was mainly the consequence of an increased tHb, but in the remaining subjects the change was the result of a decrease in the plasma volume. After the initial epo "boosting" period the assessment of tHb could not detect epo injections in 50% of the subjects in the remaining "maintenance" period. In our opinion the variability observed over time when assessing tHb is not justifiable in an anti-doping setting.

PEGylation promotes hemoglobin tetramer dissociation.

Hemoglobin conjugated with poly(ethylene glycol) (PEG) acts as an oxygen carrier free in plasma, substituting red blood cells in supplementing oxygen in hypo-oxygenation pathologies. Given the complexity of oxygen delivery controls, subtle structural and functional differences in PEGylated hemoglobins might be associated with distinct physiological responses and, potentially, adverse effects. We have compared hemoglobin PEGylated under anaerobic conditions, called PEG-Hb(deoxy), with hemoglobin PEGylated under aerobic conditions, called PEG-Hb(oxy), a product that mimics Hemospan, produced by Sangart, Inc. SDS PAGE and MALDI-TOF analyses demonstrated that PEG conjugation yields products characterized by a broad distribution of PEG/hemoglobin ratios. The elution profiles in size-exclusion chromatography indicate that both products exhibit a more homogeneous distribution of molecular weight/hydrodynamic volume under deoxy conditions and at higher concentrations. PEG-Hb(oxy) shows high oxygen affinity, low modulation of allosteric effectors, almost no cooperativity, a fast and monophasic CO binding, and a limited dependence of functional properties on concentration, whereas PEG-Hb(deoxy) exhibits oxygen binding curves that significantly depend on protein concentration, and a slow CO binding, similar to native hemoglobin. PEGylated CO-hemoglobins, probed by flash photolysis, exhibited a lower amplitude for the geminate rebinding phase with respect to native hemoglobin and a negligible T state bimolecular CO rebinding phase. These findings are explained by an increased dissociation of PEGylated hemoglobins into dimers and perturbed T and R states with decreased quaternary transition rates. These features are more pronounced for PEG-Hb(oxy) than PEG-Hb(deoxy). The detected heterogeneity might be a source of adverse effects when PEGylated Hbs are used as blood substitutes.

[A nondestructive method to measure the oxygen binding and releasing properties of biodegradable polymers microcapsules intented for blood substitutes].

P50 is an important parameter reflecting the binding and releasing oxygen properties of blood substitutes. In this study, based on the strong penetrating property of near infrared light and the mechanism involved in the pulsatile oxygen meter in clinic as well as on the ability for penetrating biodegradable polymers and detecting bovine hemoglobin encapsulated within the microcapsules, we have made an airproof and equilibrium apparatus to measure oxygen saturation and oxygen partial pressure. Subsequently, we have obtained the oxygen dissociation curve and P50 of the microcapsules loaded bovine hemoglobin in the light of oxyHemoglobin and deoxyHemoglobin with different spectrum in the near infrared region. The above-mentioned apparatus and method are not destructive to the microcapsules, and the process is simple and nondestructive. So it is practical to take in-situ measurements of the oxygen binding and releasing property of biodegradable polymer microcapsules intented for the blood substitute.

A unified approach for assessing agreement for continuous and categorical data.

This paper proposes several Concordance Correlation Coefficient (CCC) indices to measure the agreement among k raters, with each rater having multiple (m) readings from each of the n subjects for continuous and categorical data. In addition, for normal data, this paper also proposes the coverage probability (CP) and total deviation index (TDI). Those indices are used to measure intra, inter and total agreement among all raters. Intra-rater indices are used to measure the agreement among the multiple readings from the same rater. Inter-rater indices are used to measure the agreement among different raters based on the average of multiple readings. Total-rater indices are used to measure the agreement among different raters based on individual readings. In addition to the agreement, the paper also assess intra, inter, and total precision and accuracy. Through a two-way mixed model, all CCC, precision and accuracy, TDI, and CP indices are expressed as functions of variance components, and GEE method is used to obtain the estimates and perform inferences for all the functions of variance components. Each of previous proposed approaches for assessing agreement becomes one of the special case of the proposed approach. For continuous data, when m approaches infinity, the proposed estimates reduce to the agreement indices proposed by Barnhart et al. (2005). When m = 1, the proposed estimate reduces to the ICC proposed by Carrasco and Jover (2003). When m = 1, the proposed estimate also reduces to the OCCC proposed by Lin (1989), King and Chinchilli (2001a) and Barnhart et al. (2002). When m = 1 and k = 2, the proposed estimate reduces to the original CCC proposed by Lin (1989). For categorical data, when k = 2 and m = 1, the proposed estimate and its associated inference reduce to the kappa for binary data and weighted kappa with squared weight for ordinal data.

Electrophoretic, size-exclusion high-performance liquid chromatography and liquid chromatography-electrospray ionization ion trap mass spectrometric detection of hemoglobin-based oxygen carriers.

Hemoglobin-based oxygen carriers (HBOCs) are blood substitutes based on hemoglobin of either bovine or human origin and they can potentially be misused in elite sports to improve endurance performance. Recently, three methods have been proposed in doping control analysis to allow HBOCs screening and identification by application of electrophoresis, size-exclusion chromatography coupled with HPLC and LC coupled with tandem mass spectrometry (LC/MSMS). In view of the Athens 2004 Olympic Games, modifications were introduced in order to increase the specificity of these methods. The sample preparation protocols of the electrophoretic and SEC-HPLC methods were modified with the introduction of sequential ultra filtration steps to remove all heme containing material below 100 kDa, thus leaving only HBOCs material for analysis. Furthermore, a modification of the LC/MSMS methodology was introduced to allow full scan MS-MS spectra of peptide segments arising from the tryptic digestion of bovine HBOCs. These relatively simple methodological modifications have major impact, as far as time and cost effectiveness is concerned in doping control procedures, because they provide a useful tool in order to identify which suspect samples from the initial visual screening are due to hemolysis and exclude them from further analysis.

Conjugation of methoxypolyethylene glycol to the surface of bovine red blood cells.

Methoxypolyethylene glycol (mPEG) covalently bound to the surface of human red blood cells (hRBCs) has been shown to decrease immunological recognition of hRBC surface antigens (Bradley et al., 2002). However, there is an increasing shortage of hRBC donations, thus making hRBCs scarce and expensive (Davey, 2004; Riess, 2001). The goal of this study is to similarly PEGylate the surface of bovine RBCs (bRBCs) with the aim of reducing the demand on human blood donations needed for blood transfusions. This study investigates the feasibility of modifying the surface of bRBCs with the succinimidyl ester of methoxypolyethylene glycol propionic acid (SPA-mPEG) for use as a potential blood substitute. The oxygen binding affinity of PEGylated bRBCs was moderately increased with increasing initial SPA-mPEG concentrations up to 4 mM when reacted with bRBCs at a hematocrit of 12%. Oxygen transport simulations verified that SPA-mPEG conjugated bRBCs could still transport oxygen to pancreatic islet tissues even under extreme conditions. PEGylated bRBCs reconstituted to a hematocrit of 40% exhibited viscosities on the order of approximately 3 cp, similar to hRBCs at the same hematocrit. Taken together, the results of this study demonstrate the success of PEGylating bRBCs to yield modified cells with oxygen binding, transport and flow properties similar to that of hRBCs.

Arenicola marina extracellular hemoglobin: a new promising blood substitute.

The need to develop a blood substitute is now urgent because of the increasing concern over Europe's BSE outbreak and the worldwide HIV/AIDS epidemic, which have cut blood supplies. Extracellular soluble hemoglobin has long been studied for its possible use as a safe and effective alternative to blood transfusion, but this has met with little success. Clinical trials have revealed undesirable side effects-oxidative damage and vasoconstriction-that hamper the application of cell-free hemoglobin as a blood substitute. We have addressed these problems and have found a new promising extracellular blood substitute: the natural giant extracellular polymeric hemoglobin of the polychaete annelid Arenicola marina. Here we show that it is less likely to cause immunogenic response; its functional and structural properties should prevent the side effects often associated with the administration of extracellular hemoglobin. Moreover, its intrinsic properties are of interest for other therapeutic applications often associated with hemorrhagic shock (ischemia reperfusion, treatment of septic shock and for organ preservation prior to transplantation). Moreover, using natural hemoglobin is particularly useful since recombinant DNA techniques could be used to express the protein in large quantities.