A biodegradable polymer scaffold for delivery of osteotropic factors.

Despite discoveries and developments in osteotropic factors, therapies exploiting these macromolecules have been limited due to a lack of suitable delivery vehicles and three dimensional (3D) scaffolds that promote bone regeneration. To address this limitation, an emulsion freeze-drying process was developed to fabricate biodegradable scaffolds with controlled microarchitecture, and the ability to incorporate and deliver bioactive macromolecules for bone regeneration. The effect of median pore size and protein loading on protein release kinetics was investigated using scaffolds with different protein loading and median pore sizes ranging from 7 to 70 microm. Graphs of protein release from scaffolds showed an initial burst followed by a slower sustained release. Release kinetics were characterized using an unsteady-state, diffusion-controlled model with an effective diffusivity that took tortuosity (tau) and partition coefficient for protein adsorption (Kp) onto the scaffold walls into account. Tortuosity and partition coefficient significantly reduced the protein diffusivity by a factor of 41 +/- 43 and 105 +/- 51 for 60 and 30-microm median pore-sized scaffolds, respectively. The activity of the protein released from these scaffolds was demonstrated by delivering rhBMP 2 and [A-4] (an amelogenin derived polypeptide) proteins from the scaffold and regenerating bone in a rat ectopic bone induction assay [Whang et al. J Biomed Mater Res 1998;42:491-9, Veis et al. J Bone Mineral Res, Submitted].

Oxygenation of the cat primary visual cortex.

Tissue PO2 was measured in the primary visual cortex of anesthetized, artificially ventilated normovolemic cats to examine tissue oxygenation with respect to depth. The method utilized 1) a chamber designed to maintain cerebrospinal fluid pressure and prevent ambient PO2 from influencing the brain, 2) a microelectrode capable of recording electrical activity as well as local PO2, and 3) recordings primarily during electrode withdrawal from the cortex rather than during penetrations. Local peaks in the PO2 profiles were consistent with the presence of numerous vessels. Excluding the superficial 200 microm of the cortex, in which the ambient PO2 may have influenced tissue PO2, there was a slight decrease (4.9 Torr/mm cortex) in PO2 as a function of depth. After all depths and cats were weighted equally, the average PO2 in six cats was 12.8 Torr, with approximately one-half of the values being

Perfluorocarbon emulsion improves oxygenation of the cat primary visual cortex.

Tissue PO2 was measured in the primary visual cortex of anesthetized, artificially ventilated, normovolemic cats to evaluate the effect of small doses [1 g perfluorocarbon (PFC)/kg] of a PFC emulsion (1 g PFC/1.1 ml emulsion; Alliance Pharmaceutical, San Diego, CA) on brain oxygenation. The change in tissue PO2 (DeltaPO2), resulting from briefly changing the respiratory gas from room air to 100% oxygen, was measured before and after intravenous infusion of the emulsion. Before emulsion, DeltaPO2 was 51.1 +/- 45.6 Torr (n = 8 cats). Increases in DeltaPO2 of 34.0 +/- 26.1 (SD) % (n = 8) and 16. 3 +/- 8.4% (n = 6) were observed after the first and second emulsion infusions, respectively. The further increase in DeltaPO2 after the third dose (7.9 +/- 10.5%; n = 7) was not statistically significant. The observed increases in tissue oxygenation as a result of the PFC infusions appear to be the result of enhanced oxygen transport to the tissue.

Isovolemic hemodilution increases retinal tissue oxygen tension.

BACKGROUND: Therapeutic isovolemic hemodilution has been reported to improve blood flow and oxygen delivery. Few reliable measurements have been made, however, showing the effect of hemodilution on tissue oxygen tension. METHODS: We measured retinal oxygen tension during experimental isovolemic hemodilution in normal cats. Polarographic oxygen microelectrodes were placed in the vitreous humor within 100-200 microns of the retinal surface. RESULTS: Tissue oxygen tension increased initially during isovolemic hemodilution to a maximum approximately 50% above baseline at approximately two thirds of the original hematocrit level. Hemodilution beyond this point to lower hematocrits caused a steady decline in tissue oxygen tension. Cardiac output measured in one cat undergoing isovolemic hemodilution increased as hematocrit was lowered, but the cardiac erythrocyte flux actually decreased steadily. CONCLUSION: The observed increase in tissue oxygen tension with hemodilution appears to be explained by a lesser reduction in capillary than in systemic hematocrit, coupled with an increased capillary blood flow. The increase in tissue oxygen tension we observed could in part explain the clinically beneficial effects of hemodilution.

Nonlinear viscoelasticity and the thrombelastograph: 1. Studies on bovine plasma clots.

To study the possible role of nonlinear viscoelastic effects in the thrombelastograph (TEG), clotting of bovine plasma was studied by both thrombelastography and with a controlled strain rheometer. Clot rheology is dominated by elastic effects at frequencies of interest. There is a well-defined regime of linear elasticity for strains less than around 2%, while at larger strains the clots show significant strain hardening. Oscillatory shear applied during clotting has little effect on the resulting clot provided that the strain is less than 2%, but leads to substantial weakening of clots formed at larger strains. The TEG operates within the regime of nonlinear elasticity, significantly obscuring the interpretation of TEG amplitude in terms of an elastic modulus. Comparing the results of standard TEG experiments with those conducted with a modified TEG, having no oscillation during clotting, shows that deformation during standard thrombelastography leads to weaker clots than are produced under quiescent conditions.

Retinal tissue oxygen tension in normoxic cats under enflurane anesthesia.

PURPOSE: General anesthesia reduces systemic blood pressure and, thus, ocular perfusion pressure (at constant intraocular pressure). Whether this reduction in ocular perfusion pressure produces retinal hypoxia is unknown. To answer this question, the authors measured inner retinal oxygen tension in cats under general enflurane anesthesia at three clinically relevant levels of anesthesia under normoxic conditions. METHODS: Polarographic oxygen microelectrodes were used to measure inner retinal oxygen tension in cats under enflurane anesthesia at 21% inspired oxygen tension. Measurements were made in the preretinal vitreous body within 100 to 200 microns of the internal limiting membrane of the retina. Three levels of enflurane anesthesia were used: 1.2%, 2.4%, and 3.6%, corresponding to 0.5, 1.0, and 1.5 minimal alveolar concentration. Intraocular pressure of the cats was maintained at a constant normal level throughout the experiments. RESULTS: Under normoxic conditions, inner retinal oxygen tension remained unchanged or increased slightly as ocular perfusion pressure decreased with deeper levels of enflurane anesthesia. CONCLUSION: Commonly used surgical levels of enflurane general anesthesia do not cause hypoxia of the inner retina in cats breathing 21% inspired oxygen. This may be the result of preservation of retinal vascular autoregulation under enflurane anesthesia, retinal vasodilatation secondary to a direct smooth muscle relaxing effect of enflurane, or decreased retinal oxygen use under enflurane anesthesia.

Variability in measurement of central retinal artery velocity using color Doppler imaging.

The purpose of our study was to test the reproducibility of the central retinal artery velocity measurements as made by color Doppler imaging and pulsed Doppler spectral tracings and to define the optimal location for placement of the sample volume. We used the Siemens Quantum (Q 2000) with a 7.5 MHz linear array transducer to identify the central retinal artery and record its velocity. Eleven eyes from 11 normal subjects were evaluated for the peak systolic central retinal artery velocity in two positions, the first posteriorly in the optic nerve at an average of 3.56 mm from the surface of the optic disc and the second at an average of 1.76 mm from the surface of the optic disc. We then used a color-flow threshold to identify and record the point of maximal velocity. The range of systolic velocities in the 11 eyes was from 5.85 to 22.51 cm/sec. The peak systolic velocity posteriorly in the optic nerve averaged 8.16 cm/sec, whereas near the surface of the optic disc the velocity averaged 13.89 cm/sec (70.2% higher). Using the color flow threshold method, the maximal velocity was located at an average of 1.98 mm from the surface of the optic disc. Our data show that large differences exist in measured central retinal artery velocity that depend on the location of the measurement, and that color-flow thresholding is valuable in locating the optimal location for pulsed Doppler spectral recording.

Oxygen consumption in the inner and outer retina of the cat.

PURPOSE: Oxygen consumption rate (QO2) was determined in the outer and inner halves of the cat retina in dark and light adaptation. METHODS: Double-barreled oxygen microelectrodes were used to measure oxygen tension (PO2) across the retina of anesthetized cats while the single retinal artery supplying that area was occluded. During the measurements of these PO2 profiles, the cats were ventilated with 100% O2. Retinal PO2 profiles were fitted to a diffusion model, and inner and outer retinal QO2s were determined from the fitted parameters. RESULTS: A four-layer model, in which two layers consumed oxygen, fitted the data well. One consuming layer corresponded to the photoreceptor inner segments, as in previous studies, and a single region of uniform consumption was used to describe the profile in the inner half of the retina. Under dark-adapted conditions, outer and inner retinal QO2 were 3.9 +/- 2.8 and 3.5 +/- 1.7 ml O2/(100 g.min) (mean +/- SD; 9 cats), respectively. With steady illumination, outer retinal (photoreceptor) QO2 decreased to 1.4 +/- 0.9 ml O2/(100 g.min), but inner retinal QO2 remained unchanged at 3.7 +/- 1.5 ml O2/(100 g.min) (5 cats). CONCLUSIONS: The total QO2 of the inner retina was found to be the same as that of the dark-adapted outer retina. Oxygen use was distributed uniformly throughout the inner retina but was confined to the photoreceptor inner segments, which occupied approximately 20% of the thickness of the outer retina. Steady illumination had no effect on inner retinal QO2.

Enhanced oxygen delivery induced by perfluorocarbon emulsions in capillary tube oxygenators.

Previous studies showed that a new generation of perfluorocarbon (PFC) emulsions increased tissue PO2 in the cat retina to a degree that could not be explained by the small increase in arterial O2 content seen after the infusion of low doses of 1 g PFC/kg body weight. It seems that increased O2 delivery at the tissue level after PFC infusion is caused by a local effect in the microcirculation. The authors studies this effect in vitro at steady state in a closed loop circuit, consisting of one of two types of capillary tube oxygenators, deoxygenator(s), a reservoir bag filled with anticoagulated bovine blood or saline (control), and a roller pump, to see if the addition of PFC would have an effect on the PO2 difference (delta PO2) across the capillary tube membrane oxygenator at a blood flow rate of 3 l/min. Perfluorocarbon was added in three incremental doses, each giving about 0.7 vol% of PFC. The delta PO2 across the oxygenator was measured before and after each dose. The mean percent increases in delta PO2 in blood for two types of oxygenators were 19.2 +/- 8% (mean +/- SD, n = 6, P = 0.002) and 9.9 +/- 4% (n = 3, P = 0.05), respectively, whereas the mean percent change in delta PO2 in saline was -4.9 +/- 2% (n = 2, P = 0.2). Inlet PO2s to the oxygenator were only minimally increased. The authors conclude that O2 delivery was significantly enhanced after injection of PFC in blood in this capillary tube model. A near wall excess of PFC particles may account for the augmentation of O2 diffusion in this model.

New perfluorocarbon emulsion improves tissue oxygenation in cat retina.

We have studied the conditions under which a perfluorocarbon emulsion of perfluorooctyl bromide (PFOB; Alliance Pharmaceutical, San Diego, CA) enhances tissue O2 delivery. Measurements of retinal tissue O2 tension (PO2) were made in anesthetized, artificially respirated, dark-adapted, normovolemic cats before, during, and after the infusion of three successive doses of 1 g PFOB/kg body wt each. There was little immediate effect of the infusion on the tissue PO2 when the cats were breathing room air, but the mean increase in tissue PO2 during 100% O2 breathing was 60 +/- 9% (SE; n = 8 cats) greater after infusion of 1 g PFOB/kg and approximately 136% greater after 3 g PFOB/kg. Similar infusions of the emulsifying medium alone had negligible effects on tissue PO2. These results suggest that PFOB emulsion may be clinically useful in treating tissue hypoxia in normovolemic patients breathing O2-enriched air.

Spatial variation of aortic wall oxygen diffusion coefficient from transient polarographic measurements.

Polarographic current transients following a voltage step (turn-on transient) were measured with bare cathodes (25 microns diameter) and shallowly recessed oxygen microelectrodes (< 5 microns diameter). Except for the initial part of the current transient, the experimental measurements were in excellent agreement with simple models in the literature, which predict an inverse relationship with square root of t. Turn-on transients were measured in aqueous solutions with known physical properties, and in aortic wall tissue from three different species (n = 6 rabbits, n = 3 dogs, and n = 1 miniature pig). Oxygen diffusion coefficients (D) were determined in vitro by comparing time constants measured by the same microelectrode in saline and in strips of aortic wall tissue at 37 degrees C. On the inner side (endothelium and intima) of the aorta, D averaged (+/- S.E.) 7.0 (+/- 0.8) x 10(-6) cm2/s in 6 rabbits, 6.4 (+/- 1.0) x 10(-6) cm2/s in 3 dogs, and was 4.6 x 10(-6) cm2/s in the pig. On the adventitial side, D was 9.5 x 10(-6) cm2/s in 1 rabbit, 11.4 (+/- 1.2) x 10(-6) cm2/s in 3 dogs, and 8.1 x 10(-6) cm2/s in the pig. For every aortic strip on which D was measured from both sides, D for the inner wall was always lower, overall by a little more than one third (p < 0.001). The lower D on the endothelial side may limit oxygen transport to the vascular wall and play a role in atherogenesis.

Mathematical models of the spatial distribution of retinal oxygen tension and consumption, including changes upon illumination.

To better understand oxygen utilization by the retina, a mathematical model of oxygen diffusion and consumption in the cat outer, avascular retina was developed by analyzing previously recorded profiles of oxygen tension (PO2) as a function of retinal depth. Simple diffusion modelling of the oxygen distribution through the outer retina is possible because the PO2 depends only on diffusion from the choroidal and retinal circulations and on consumption within the tissue. Several different models were evaluated in order to determine the best one from the standpoints of their ability to represent the data and to agree with physiological reality. For the steady state one-dimensional diffusion model adopted (the special three-layer diffusion model), oxygen consumption was constant through the middle layer and zero in the layers near the choroid and near the inner retina. On the average, the oxygen consuming layer, as found by nonlinear regression for each profile, extended from about 75% to 85% of the retinal depth from the vitreous. This is a narrow band through the mid-region of the photoreceptors. Oxygen consumption of the entire avascular retina, determined from fitting eight PO2 profiles measured in light-adapted retinas, averaged 2.7 ml O2(STP)/(100 g tissue.min), while the value determined from fitting thirty-two PO2 profiles measured in dark-adapted retinas averaged 4.4 ml O2(STP)/(100 g tissue.min). Consumption in the light was thus only 60% of that in the dark. This suggests that the outer retina is at greater risk of hypoxic injury in the dark than in the light, a finding of considerable clinical significance.

Spatial variation of the local tissue oxygen diffusion coefficient measured in situ in the cat retina and cornea.

A method for measuring the local oxygen diffusion coefficient (D) in an intact tissue, in situ, in a living cat is described. Values of D were calculated from nonlinear regression analysis of the polarographic (turn-on) transients using a semi-empirical model for the retina and a theoretical one for the cornea. Two types of microelectrodes were employed: in the retina, ones with extremely short recesses; and in the cornea, bare metal needles. The local D in the cat retina was practically homogeneous with a mean of 1.97 +/- 0.11.10(-5) cm2/s, at its body temperature of 37-38 degrees C, 70.6 +/- 3.3 percent of that in isotonic saline at 37 degrees C. In the cat corneal stroma, at its normal temperature in situ of 33 degrees C, D was also virtually homogeneous with a mean of 1.19 +/- 0.20.10(-5) cm2/s, 42.8 +/- 7.3 percent of that in isotonic saline at 37 degrees C.

Chinese herbal medicine increases tissue oxygen tension.

An herbal medicine widely used in China, anisodamine, was investigated to determine its effect on the tissue PO2 of an important neural tissue, the retina. The anisodamine was injected intravenously over 10 min into anesthetized cats. Although it reduced mean blood pressure an average of 26%, anisodamine simultaneously increased tissue PO2 an average of 20%. It may therefore be useful in treating clinical conditions thought to be secondary to tissue hypoxia, such as diabetic retinopathy and sickle cell crises.

Tissue oxygen uptake from the atmosphere by a new, noninvasive polarographic technique with application to corneal metabolism.

A standard Clark electrode has been modified to continuously monitor the PO2 in a thin, disk shaped reservoir membrane mounted on the electrode membrane surface. In vitro tests were conducted to determine the proportionality constant between the rate of change of reservoir PO2 and the flux of O2 out of the reservoir. The device was then used to determine the corneal O2 uptake on proptosed eyes of previously sacrificed rabbits. Our average measured uptake at 20 degrees C, 1.7 microliter/cm2.hr, agrees with the value 2.3 microliters/cm2.hr calculated from a diffusion analysis of the cornea utilizing literature values of the parameters at 37 degrees C for the three component layers of the cornea when they are adjusted for temperature to 20 degrees C.

Effect of peripheral cryocoagulation on ocular blood flow.

Using the labeled microsphere impaction technique, we studied the effect of peripheral panretinal transscleral cryocoagulation in pigmented rabbits. One eye of each animal received peripheral cryocoagulation, and the fellow eye of the same animal was used as the control. Thirty-two animals received 40 exposures of peripheral cryocoagulation; twenty-six rabbits received 20 exposures. After 40 exposures of cryocoagulation, choroidal-retinal blood flow was significantly decreased when measured at one to two hours, 24 hours, and two weeks; normal flow recovered by four weeks. After 20 exposures of cryocoagulation, there was a significant reduction in choroidal-retinal blood flow at the earliest measurement time (one to two hours) only. The iris-ciliary body blood flow also was substantially decreased only at the earliest measurement time (one to two hours) and only after 40 exposures.

Tumour blood flow following local ultrasound heating computed from thermal clearance curves.

Thermal clearance curves following termination of ultrasound-induced hyperthermia in human mammary carcinomas implanted into the flanks of nude rats were studied. They were found to be monoexponential in form, both with and without blood flow. From the difference between the inverse time constants with and without flow, the tumour blood flow rate could be calculated. Blood flow was found to increase with very short exposure times at the therapeutic hyperthermia temperature and subsequently decrease as the exposure time increased. A higher therapeutic hyperthermia temperature augmented this effect.

Oxygen tension changes in the outer vascular wall supplied by vasa vasorum following adenosine and epinephrine.

We measured blood vessel wall oxygen tension (PO2) in vivo in the tissue and on the surface of dog thoracic aortas and femoral arteries. The overall mean tissue PO2 in the outer vascular wall supplied by vasa vasorum for both blood vessels was 49.4 +/- 2.6 (SE) Torr for 42 sites in 8 dogs. Changes in PO2 were measured after intravenous injections of adenosine and epinephrine. There was some tendency for the PO2 to decrease with epinephrine for both vessels. With adenosine the thoracic aorta wall tissue PO2 increased significantly (+23.2 +/- 6.0 Torr, p less than 0.025), while the femoral artery did not. One possible interpretation of these results is that the vasa vasorum of the thoracic aorta are capable of changing blood flow and oxygen supply to the outer wall to a greater degree than the femoral artery.

Hyaluronate increases intraocular pressure when used in cataract extraction.

A prospective randomised study of 26 eyes (24 patients) following uncomplicated intracapsular cataract surgery was carried out with three groups in which different agents were used to re-form the anterior chamber: air, seven eyes; sodium hyaluronate, seven eyes; and a combination of hyaluronate plus systemic acetazolamide, 12 eyes. The intraocular pressure (IOP) was measured by Goldmann applanation tonometry 16 hours before surgery and every eight hours after surgery for 72 hours. The IOP doubled in the two hyaluronate-treated groups for the first day following cataract surgery. By contrast, the IOP in the group receiving air initially decreased slightly. Throughout the study the effect of using acetazolamide was never statistically significant. After the first day and a half the differences in IOP among the three treatment groups disappeared, and the pressures returned to their preoperative values.