Biomimetic calcium phosphate/polyelectrolyte multilayer coatings for sequential delivery of multiple biological factors.

Combinations of growth factors synergistically enhance tissue regeneration, but typically require sequential, rather than co-delivery from biomaterials for maximum efficacy. Polyelectrolyte multilayer (PEM) coatings can deliver multiple factors without loss of activity; however, sequential delivery from PEM has been limited due to interlayer diffusion that results in co-delivery of the factors. This study shows that addition of a biomimetic calcium phosphate (bCaP) barrier layer to a PEM coating effectively prevents interlayer diffusion and enables sequential delivery of two different biomolecules via direct cell access. A simulated body fluid method was used to deposit a layer of bCaP followed by 30 bilayers of PEM made with poly-l-Lysine (+) and poly l-Glutamic acid (-) (bCaP-PEM). Measurements of MC3T3-E1 proliferation and viability over time on bCaP-PEM were used to demonstrate the sequential delivery kinetics of a proliferative factor [fibroblast growth factor-2 (FGF-2)] followed by a cytotoxic factor (antimycin A, AntiA). FGF-2 and AntiA both retained their bioactivity within bCaP-PEM, yet no release of FGF-2 or AntiA from bCaP-PEM was observed when cells were absent indicating a cell-mediated, local delivery process. This coating technique is useful for a variety of applications that would benefit from highly localized, sequential delivery of multiple biomolecules governed by cell initiated degradation that avoids off-target effects associated with diffusion-based release. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1500-1509, 2017.

[Bovine hemoglobin. HBOC-201 causes a reduction of the oxygen partial pressure in poststenotic skeletal muscle]. / Bovines Hämoglobin. HBOC-201 verhindert eine Reduktion des Sauerstoffpartialdrucks im poststenotischen Skelettmuskel.

UNLABELLED: We investigated the effects of ultrapurified polymerized bovine hemoglobin (HBOC-201) on skeletal muscle tissue oxygen tension when applied before establishment of a nearly complete arterial stenosis. METHODS: Twelve foxhounds were anaesthetized IV and mechanically ventilated with 30% oxygen in air. Catheters were inserted into the right femoral artery and vein for measurements of haemodynamic parameters and blood-gas sampling. Arterial blood flow of the left popliteal artery was measured by an electromagnetic flow probe. Skeletal muscle tissue oxygen tension (tpo2) was measured in the left gastrocnemic muscle using a stepwise-driven polarographic needle probe, creating histograms from 200 single tpO2 measurements. Following isovolaemic haemodilution with Ringer's solution to a target haematocrit of 20%, the animals were randomly assigned to receive either 200 ml of predonated fresh blood (group 1) or 200 ml of HBOC-201 (MW 32,000-500,000; Hb 13 +/- 1 g-dl-1, group 2). After a 15-min stabilization period, a 95% artificial stenosis of the left popliteal artery was established. While animals of group 1 received two applications of 200 ml 6% hetastarch (HES, 200,000; 0.5), animals of group 2 received 200 ml Ringer's solution 45 and 75 min after establishment of the arterial stenosis, respectively. Variables were measured at baseline, after haemodilution and application of the respective compound, and 30, 60 and 90 min after establishment of the stenosis. RESULTS: Demographic data, muscle temperature and arterial blood gases did not differ between groups. With the exception of a higher mean pulmonary artery pressure in HBOC-201-treated animals, haemodynamics did not differ between groups. In both groups oxygen delivery and oxygen consumption of the muscle decreased in parallel to the decreasing blood flow during arterial stenosis. In contrast, oxygen extraction ratio increased after infusion of HBOC-201 and remained unchanged during stenosis (P < 0.05). In group 1, the tpO2 decreased during stenosis when compared to baseline (P < 0.001) and remained decreased after administration of HES. In contrast, administration of 200 ml of HBOC-201 before establishment of the arterial stenosis sustained the tpO2 values at nearly baseline levels during stenosis. Skeletal muscle tissue oxygen tension was higher after HBOC-201 infusion during stenosis when compared to HES infusion (P < 0.001). CONCLUSION: These data suggest that haemoglobin solutions can reach poststenotic tissues. The increased oxygen extraction after application of HBOC-201 is associated with improved skeletal muscle oxygen tension during severe arterial stenosis.

A phase I/II study of polymerized bovine hemoglobin in adult patients with sickle cell disease not in crisis at the time of study.

BACKGROUND: The painful episodes of sickle cell disease (SCD) involve vaso-occlusion and impaired oxygen delivery. HBOC-201, a hemoglobin-based oxygen carrier, has been shown to support oxygen delivery in animal studies and to be safe and well tolerated in normal human volunteers. Therefore, we speculated that it might have a therapeutic role in SCD. METHODS: Eighteen adults with SCD who were asymptomatic at the time of study were enrolled in a Phase I/II single-blind, placebo-controlled, dose-escalation study of HBOC-201. The primary purpose was to assess the safety of the material in this patient population. In addition, as a surrogate marker of efficacy, each subject underwent a variety of exercise tests before and after HBOC-201 was given. RESULTS: All HBOC-201 infusions were well tolerated by the study subjects and no evidence of toxicity was noted. In addition, there was a significant difference in heart rate response to the identical aerobic exercise workload when the study subjects who received HBOC-201 were compared to the subjects who received placebo (p = 0.0061). CONCLUSIONS: HBOC-201 was safely administered to patients with SCD who were not in crisis at the time of study. Furthermore, following infusion of the study material, subjects with SCD performed the identical aerobic exercise-induced workload with an increase in heart rate that was significantly less than the increase observed in the subjects who received an infusion of the saline placebo. These safety and surrogate efficacy data support the notion that HBOC-201 could have efficacy as a treatment for the vasoocclusive episodes of SCD.

Bovine hemoglobin increases skeletal muscle oxygenation during 95% artificial arterial stenosis.

BACKGROUND: This study investigates the effect of a stroma-free ultrapurified bovine hemoglobin solution (HBOC) on skeletal muscle tissue oxygenation in comparison with hetastarch during nearly complete arterial stenosis. METHODS: Fourteen foxhounds were intravenously anesthetized and mechanically ventilated with 30% oxygen in air. Catheters were inserted into the right femoral artery and vein for measurements of hemodynamic parameters and blood gas sampling. Arterial blood flow of the left popliteal artery was measured by means of an electromagnetic flow probe. Skeletal muscle tissue oxygen tension (tpO2) was measured in the left gastrocnemius muscle by using a stepwise driven polarographic needle probe creating histograms from 200 single tpO2 measurements. After isovolemic hemodilution with Ringer's lactate solution to a hematocrit of 25%, a 95% artificial stenosis of the popliteal artery was established. The animals then randomly received two applications of either 50 ml HBOC (molecular weight, 32,000 to 500,000; hemoglobin, 13 +/- 1 gm/dl-1) or 200 ml 6% hetastarch 200,000/0.5. Variables were measured at baseline, after hemodilution, 30 minutes after stenosis, and 15 minutes after two applications of the respective compound. RESULTS: Demographic data, muscle temperature, and arterial blood gases did not differ between groups. With the exception of higher mean arterial and mean pulmonary artery pressures in HBOC-treated animals, hemodynamics did not differ between groups. In both groups oxygen delivery and oxygen consumption of the muscle decreased in parallel to the decreasing blood flow during arterial stenosis. In contrast, oxygen extraction ratio increased after infusion of HBOC and was higher after the second application when compared with hetastarch-treated animals (p < 0.05). During stenosis tpO2 was decreased in both groups when compared with baseline (p < 0.001). Mean tpO2 remained at decreased levels after administration of hetastarch but increased to nearly baseline values after HBOC treatment (p < 0.001). CONCLUSIONS: The data suggest that increased oxygen extraction in the HBOC group is associated with improved skeletal muscle tissue oxygenation during severe arterial stenosis.

Physiology and pharmacokinetics of a novel hemoglobin-based oxygen carrier in humans.

OBJECTIVE: To evaluate the physiology and pharmacokinetics of a novel hemoglobin-based oxygen carrier of bovine origin. DESIGN: Randomized, single-blind, placebo-controlled, dose-escalation study. SETTING: The Upjohn Research Clinics (Kalamazoo, MI). SUBJECTS: Normal healthy adult men between the ages of 18 and 45 yrs. There were 18 subjects who received active treatment and 23 controls. INTERVENTIONS: All subjects had phlebotomy of 15% of blood volume (performed in <15 mins) followed by isovolemic hemodilution (3:1, Ringer's lactate to the volume of whole blood removed) over a 90-min period, and either active drug (polymerized bovine hemoglobin) or a control infusion of lactated Ringer's solution (each infusion given over a total of 4.3 hrs). The subjects randomized to active treatment received a loading dose and a continuous infusion of polymerized bovine hemoglobin for a total dose of 16.5, 24.1, 30.2, 38.0, or 45.0 g. All subjects had an indwelling radial artery catheter (for blood pressure and arterial blood gas measurements), determination of cardiac function (by impedance plethysmography), serial pulmonary function tests (spirometry and diffusion capacity), and metabolic cart measurements. MEASUREMENTS AND MAIN RESULTS: Pharmacokinetics of the plasma bovine hemoglobin demonstrated that the elimination of the hemoglobin-based oxygen carrier was a linear, first-order process and that there was no renal excretion. Peak plasma concentrations were between 1 to 2 g/dL and plasma half-life approached 20 hrs at the highest doses given. Diffusion capacity of oxygen was increased up to 20% above baseline in the 38.0 and 45.0 g groups in comparison with controls (approximately 14% below baseline) between 2 and 24 hrs after the infusion (p < .01). Other pulmonary function tests and arterial blood gas measurements were unremarkable. Arterial oxygen content and oxygen delivery tended to be greater in active groups than in controls. CONCLUSIONS: The plasma concentrations of bovine hemoglobin were directly proportional to the doses administered. An increase in diffusion capacity paralleled the plasma bovine hemoglobin concentrations. Dosing of the hemoglobin-based oxygen carrier of bovine origin to a target plasma hemoglobin concentration can be achieved using pharmacokinetic principles with measurable effects on oxygen physiology.

Effects of a novel hemoglobin-based oxygen carrier on percent oxygen saturation as determined with arterial blood gas analysis and pulse oximetry.

STUDY OBJECTIVE: Hemoglobin-based oxygen carrier 201 (HBOC-201) is a polymerized hemoglobin of bovine origin being developed for use in hemorrhage during surgery or trauma. Pulse oximetry is commonly used in clinical practice to assess percent saturation of hemoglobin (Spo2). The ability to measure Spo2 in the presence of HBOC-201 will be important for the use of this compound in patient care. METHODS: We carried out a randomized, single-blind, placebo-controlled study at the Upjohn Research Clinics in Kalamazoo, Michigan, with normal, healthy adult men and women as subjects. The members of four groups of adult subjects (N=24) each received 45 g of HBOC-201 (nine each, men and women) or a control solution (Ringer's lactate) (three each, men and women). Each subject underwent phlebotomy (about 15% of estimated blood volume) followed by 3:1 hemodilution with Ringer's lactate and then either HBOC-201 or control solution. An indwelling arterial catheter in the radial artery was used for serial arterial blood gas sampling. Arterial blood gas measurements were made with a cooximeter (Instrumentation Laboratories). Fingertip pulse oximetry was used (Criticare 504-US; Criticare, Incorporated). Paired pulse oximetry and arterial blood gas sampling were made serially (at approximately hourly intervals) over 24 hours. RESULTS: The mean +/- SEM difference for Spo2 for arterial blood gas analysis compared with the pulse oximetry reading in the presence of HBOC-201 was 1.1% +/- 0.75%; in controls it was .1% +/- 0.64% (P<.001) for each) over the 24 hours after dosing. This relationship was constant despite increased concentrations of plasma hemoglobin (between 1 and 2g/dl [10 to 20 g/L]) in the HBOC-201 groups. CONCLUSION: Accurate determinations of Spo2 can be made with pulse oximetry in subjects given HBOC-201 over the normal range of Spo2.

Hematologic effects of a novel hemoglobin-based oxygen carrier in normal male and female subjects.

The objective of this study was to assess the relationship between iron metabolism and pharmacokinetics of hemoglobin-based oxygen carrier-201 (HBOC-201), a polymerized hemoglobin product of bovine origin. A randomized, single-blind, single-dose study design was used. The study was performed at the Upjohn Research Clinics in Kalamazoo, Michigan. Four groups of healthy men and women (n = 24), who either received HBOC-201 (9 men, 9 women) or a control solution (Ringer's lactate) (3 men, 3 women) participated in the study. All subjects had phlebotomy (approximately 15% blood volume) followed by 3:1 hemodilution with Ringer's lactate and an intravenous infusion of HBOC-201 (up to 45 gm or 350 ml) or control solution (Ringer's lactate). Serial arterial blood gas samples with a radial artery catheter and simultaneous pulse oximetry were done during the first 24 hours. Serial samples for serum iron, ferritin, erythropoietin, and plasma HBOC-201 levels were taken over a 1-month period. In the HBOC-201-treated groups, serum iron and ferritin levels increased. Peak serum iron and ferritin levels occurred by hours 8 (up to 220 micrograms/dl) and 48 (up to 180 ng/ml), respectively. Serum iron levels paralleled HBOC-201 concentrations. Plasma half-life of HBOC-201 was about 20 hours. Serum erythropoietin increased by twofold to sixfold over baseline (p < 0.001) at 24 hours. No urinary hemoglobin was detected in the groups with HBOC-201-treated subjects. This study demonstrates that HBOC-201 produces increases in serum iron, ferritin, and erythropoietin that closely parallel plasma levels of HBOC-201 in men and women.

Hemoglobin-based oxygen carrier preserves submaximal exercise capacity in humans.

OBJECTIVE: The objective of this study was to evaluate the exercise capacity of subjects given an autologous transfusion or a polymerized bovine hemoglobin solution to define the pharmacodynamics and pharmacokinetics of a new hemoglobin-based oxygen carrier (HBOC-201). METHODS: Six normal healthy male subjects (ages 25 to 45 years) participated in this randomized, single-blind, two-way crossover study, which took place at Upjohn Research Clinics in Kalamazoo, Mich. A radial artery catheter was inserted in each subject before serial cardiac output and pulmonary function tests and phlebotomy of 15% blood volume (750 ml plus another 250 ml for study laboratories yields 1000 ml, or about 150 gm human hemoglobin). This was followed by isovolemic hemodilution with Ringer's lactate plus an autologous blood transfusion (or HBOC-201) and 1 week later 45 gm bovine hemoglobin of HBOC-201 (or autologous transfusion). Bicycle exercise stress tests to anaerobic threshold (approximately 65% of predicted maximum aerobic capacity) were done before phlebotomy and at approximately 45 minutes after the autologous transfusion or HBOC-201 infusion. RESULTS: Subjects had similar exercise and diffusion capacity but lower lactate levels (for up to 24 hours) during HBOC-201 (which paralleled plasma HBOC-201 levels) than during autologous transfusion periods. Oxygen use (uptake) and carbon dioxide production at rest were greater during the HBOC-201 infusion than during the autologous transfusion period. The half-life of HBOC-201 was about 23 hours. CONCLUSIONS: Exercise capacity and diffusion capacity were similar after HBOC-201 and autologous transfusion. HBOC-201 resulted in greater oxygen (or uptake) and carbon dioxide production and lower lactate levels compared with autologous transfusion. Under the conditions of the study, the physiologic effects of 1 gm bovine hemoglobin of HBOC-201 were similar to 3 gm human hemoglobin from autologous transfusion.

Hemoglobin blood substitutes in extended preoperative autologous blood donation: an experimental study.

BACKGROUND: The risks of homologous blood transfusion have stimulated interest in developing possible alternatives. In this study we examined the efficacy of using a hemoglobin-derived blood substitute to augment and extend preoperative autologous blood donation. METHODS: In an ovine model, two experimental groups (n = 6 each) of animals donated either 45% or 80% or more of calculated blood volume, which was replaced with a polymerized bovine hemoglobin solution. Two control groups (n = 6 each) either did not donate or donated 45% of calculated blood volume, which was replaced with a 6% hetastarch solution. Twenty-four hours after blood donation, sheep underwent a measured surgical stress with standardized intraoperative blood loss; donated blood was reinfused. RESULTS: Extended autologous blood donation and replacement with this blood substitute were efficacious in supporting oxygen consumption; there was no systemic toxicity. After the postoperative replacement of autologous blood, animals that donated 80% or more of red cell mass exhibited overall blood conservation with a final hematocrit of 25.7% +/- 3.6%, compared with 20.5% +/- 2.0% (p < 0.05) and with 21.5% +/- 1.1% (p < 0.05) in both control groups. CONCLUSIONS: This study suggests that the benefits of a blood substitute can be combined with autologous blood donation to (1) safely increase the amount of autologous blood donated while supporting oxygen consumption, (2) avoid the need for advanced preoperative donation, and (3) decrease the need for homologous blood transfusion.

Acute effects of massive transfusion of a bovine hemoglobin blood substitute in a canine model of hemorrhagic shock.

The capability of stroma-free hemoglobin solutions to act as a plasma expander with oxygen and carbon dioxide transport properties has encouraged the idea of their possible use in settings of massive blood loss. Using a canine hemorrhagic shock model (systolic arterial pressure < or = 50 torr for 60 min), we evaluated the efficacy of an ultra-pure stroma-free bovine hemoglobin solution (PBHg) as a resuscitation fluid in hypovolemic and acidotic animals, using homologous blood (PRBC) and 10% human serum albumin (HSA) as control solutions. Following volume replacement, dogs were studied for 2 h under anesthesia and for 4 h subsequently while awake. Resuscitation with PBHg (30 +/- 3 ml/kg) was able to restore stable hemodynamics and correct acidosis to an extent comparable to that in animals treated with PRBC. Additionally, oxygen transport was maintained at a higher level than that in dogs treated with HSA. Administration of PBHg in this shock model revealed no significant cardiopulmonary toxicity or adverse effects. These short-term results suggest that PBHg may be useful for effective resuscitation after major blood loss.

Ultra-pure polymerized bovine hemoglobin blood substitute: effects on the coronary circulation.

The effects of stroma-free hemoglobin (SFHgb) on the coronary circulation remain unclear. An intact canine model utilizing intracoronary adenosine to abolish the confounding effect of autoregulation was used to study maximal myocardial oxygen delivery during progressive hemodilution with polymerized bovine SFHgb. The circumflex coronary artery was instrumented with a flow probe, hydraulic constrictor, and proximal and distal catheters for adenosine infusion and distal pressure measurement, respectively. This preparation was used to generate diastolic coronary pressure-flow relations during maximal vasodilation. Maximal coronary conductance and maximal myocardial oxygen delivery were determined in two groups of 7 dogs each following hemodilution, first with 6% hetastarch (Control), followed by further hemodilution with ultra-pure, polymerized, bovine SFHgb. After hemodilution with SFHgb, maximal coronary flow increased slightly without evidence of coronary vasoconstriction. Since hemodilution with this material increases oxygen carrying capacity, maximal oxygen delivery is greater than Control, despite the very low canine hematocrit. These findings suggest: 1) SFHgb can provide adequate oxygen delivery to the myocardium despite extreme degrees of hemodilution, and 2) in this intact model, there is no evidence of adverse coronary vasomotion.

Hemodynamic effects and oxygen transport properties of a new blood substitute in a model of massive blood replacement.

Recent concerns regarding the safety of the national blood supply have rekindled interest in the development of blood substitutes. Clinical studies have dampened the initial enthusiasm for fluorocarbon solutions as blood substitutes. The potential of hemoglobin solutions as blood substitutes has continued to stimulate investigations. However, the development of an ideal hemoglobin-derived blood substitute has eluded investigators for the past century. A persistent problem has been the inability to develop hemoglobin solutions that provide adequate oxygen and carbon dioxide exchange, while avoiding toxicity that precludes clinical safety and long-term survival. Traditionally, investigators have focused on human hemoglobin solutions. The use of outdated banked blood or pedigree human donor blood as a hemoglobin source poses continued disease transmission risks and a prohibitively limited supply. We evaluated the hemodynamic and gas transport effects of a new purified, polymerized bovine hemoglobin preparation. Bovine hemoglobin oxygen affinity is regulated by chloride ion. The concentration of chloride ions in human plasma results in excellent oxygen transport properties in a stroma-free environment. In addition, unlike human blood, bovine blood is a more disease-free hemoglobin source that is available in large supply. We exchange-transfused eight conscious sheep with this new polymerized bovine hemoglobin solution. All animals tolerated greater than or equal to 95% exchange transfusion to reach a final ovine hematocrit of 2.4 +/- 0.5% with stable hemodynamics and no clinical evidence of distress. The exchange transfusion with bovine hemoglobin polymer resulted in a final plasma hemoglobin concentration of 6.1 +/- 1.6 gm/dl, which supported oxygen consumption at baseline levels. All animals that were exchange transfused with this preparation survived long term with rapid resynthesis of ovine erythrocytes.

Ultrapure, stroma-free, polymerized bovine hemoglobin solution: evaluation of renal toxicity.

Because of recent concern about the safety of our national blood supply, there is increased interest in finding safe and effective blood substitutes. One option is the use of stroma-free hemoglobin (SFH) solutions. Recently, a SFH solution based on ultrapure, polymerized bovine hemoglobin (UPPBHg) has been shown to be effective in oxygen transport. We examined the potential renal toxicity of this material. Sprague-Dawley rats were infused with UPPBHg at doses of 25, 50, 75, and 100 ml/kg. Additional groups of rats were infused with UPPBHg at these doses with the addition of bicarbonate at a dose adequate to alkalinize the urine. Further groups of rats received UPPBHg intentionally contaminated with raw bovine blood lysate. Renal function was examined by subsequent determination of serum creatinine. UPPBHg infusion up to 50 ml/kg caused no significant change in serum creatinine; at higher doses, there was a reversible rise in creatinine at 24 hr following infusion. Addition of bicarbonate diminished the amount of reversible toxicity seen, even at doses of 100 ml/kg. In contrast, with hemolysate-contaminated UPPBHg, there were sharp increases in creatinine 24 hr after infusion of all doses tested, even at 25 ml/kg; these did not decrease significantly by 48 hr following infusion. At the higher doses tested, death occurred. These observations were not affected by simultaneous bicarbonate infusion. This study shows that UPPBHg may be administered in very large doses with only mild, reversible renal toxicity. The observation that urine alkalinization ameliorates this toxicity suggests that this may occur by hemoglobin precipitation or by a toxic effect in the renal tubules.(ABSTRACT TRUNCATED AT 250 WORDS)

Osteomyelitis of the frontal bone.

Osteomyelitis of the frontal bone and its complications remain a diagnostic and therapeutic challenge to the head and neck surgeon. This paper will deal with the etiology, pathogenesis, diagnosis, and treatment of osteomyelitis of the frontal bone, and its complications and sequelae. Special emphasis is placed on techniques for repair of forehead defects.

Silicone tracheal cannula. Update.

The Montgomery silicone tracheal cannula is a current alternative to the standard tracheotomy tube. This paper extends the 1986 report to include unusual indications for its use, complications and their management, and further recommendations on its use gathered from recent experience.

Improved case finding for pediatric hearing loss: cost analysis of school screening programs.

The majority of school hearing conservation programs employ pure tone identification audiometry as a basis of referral for further evaluation. An analysis of the cost impact of school screening programs is presented and a method of relating costs to screening program accuracy is described. This type of analysis may be used as a model for estimating cost impact and cost-benefit for pure tone audiometry and other techniques of screening for hearing loss.