Search PubMed⌕ Search

Biomedical subjects

D E Pegg

Publications and source records attributed to D E Pegg.

At least 19 recordsLinked to original sources
PubMed1998

Sensitivity of kidney perfusion protocol design to physical and physiological parameters.

The introduction and removal of cryoprotective agents (CPA) to a kidney via vascular perfusion may induce changes in cell volume that are destructive to the tubular epithelial or capillary endothelial cells as well as causing significant increases in vascular resistance that compromise the perfusion process. A network thermodynamic model of the coupled osmotic, hydrodynamic and elastic properties of the kidney was applied to evaluate the sensitivity of these critical outputs to a set of physiological and perfusion variables. Simulation results suggest that in the design of perfusion protocols for CPAs such as glycerol, it may be advantageous to: (a) select a CPA with as high a cell membrane permeability as possible; (b) increase the concentration of mannitol in the perfusate to about 200 mos/kg, beyond which there is no discernible benefit; (c) when glycerol is the CPA, limit the rate of reduction in the perfusate during removal to 30 mM/min or less; (d) limit the perfusion pressure to 20-30 mm Hg, within the practical constraints of the perfusion system; (e) increase the concentration of impermeant in the perfusate to as high as 400 mos/kg, although it is recognized that this departure from plasma-like composition might impose other problems that are not considered in this model. Further, it was observed that the vascular membrane permeability plays a relatively minor role in controlling cellular osmotic injury and vascular perfusion resistance and is therefore not a critical parameter in the perfusion design process.

Animals↗
PubMed1998

Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation.

The recent improvements in the treatment of cancer by chemo- and radiotherapy have led to a significant increase in the survival rates of patients with malignant disease, but at the expense of distressing side effects. One major problem, especially for younger patients, is that aggressive therapy destroys a significant proportion of the follicular population, which can result in either temporary or permanent infertility. Freeze-banking pieces of ovarian cortex prior to treatment is one strategy for preserving fecundity. When the patient is in remission, fertility could, theoretically, be restored by autografting the thawed tissue at the orthotopic site or by growing isolated follicles to maturity in vitro. Recent studies have found good follicular survival in frozen-thawed human ovarian tissue but to optimize the process an effective cryopreservation method needs to be developed. An essential part of such a technique is to permeate the tissue with a cryoprotectant to minimize ice formation and the extent of this equilibration is an important determinant of post-thaw cellular survival. In the current study, we have investigated the diffusion of four cryoprotective agents into human tissue at both 4 degrees C and 37 degrees C. We have also studied the effect of adding different concentrations of the non penetrating cryoprotective agent, sucrose, to the freezing media using the release of lactate dehydrogenase as a measure of its protective effect. At 4 degrees C propylene glycol and glycerol penetrated the tissue significantly slower than either ethylene glycol or dimethyl sulphoxide. At the higher temperature of 37 degrees C all four cryoprotectants penetrated at a faster rate, however concern about enhanced toxicity prevents the use of these conditions in practice. Thus, the results suggest that the best method of preparing tissue for freezing is exposure for 30 min to 1.5 M solutions of ethylene glycol or dimethyl sulphoxide at 4 degrees C; this achieved a mean tissue concentration that was almost 80% that of the bathing solution. We also report that the addition of low concentrations of sucrose to the freezing medium does not have a significant protective effect against freezing injury.

Adult↗
PubMed1997

Effects of ionizing radiation on the mechanical properties of human bone.

Allogeneic bone grafts are frequently sterilized by means of ionizing radiation. We investigated the effects of ionizing radiation on both quasistatic and impact mechanical properties of human bone. Specimens from four paired femora of four donors received doses of 29.5 kGy ("standard," frequently used by tissue banks), 94.7 kGy ("high"), or 17 kGy ("low") of ionizing radiation. Young's modulus was unchanged by any level of radiation. Radiation significantly reduced bending strength, work to fracture, and impact energy absorption; in each case, the severity of the effect increased from low to standard to high doses of radiation. Work to fracture was particularly severely degraded; specimens irradiated with the high dose absorbed only 5% of the energy of the controls. Radiation, even at relatively low doses, makes the bone more brittle and thereby reduces its energy-absorbing capacity. We suggest that because the level of radiation required to produce an acceptable level of viral inactivation (90 kGy) produces an unacceptable reduction in the mechanical integrity of the bone, low levels of radiation, sufficient to produce bacterial safety, should be used in conjunction with biological tests to ensure viral safety.

Adult↗
PubMed1997

Freeze drying of cardiac valves in preparation for cellular repopulation.

When freeze-dried cardiac valves have been implanted they remained acellular. This study is the initial step in the development of a method designed to repopulate the substance of the freeze-dried valve with fibroblasts and the lumenal surface with endothelial cells. In this scheme, the freeze-drying process performs three functions; it provides a porous matrix, it kills the donor cells, and it preserves the collagen structure and hence the mechanical strength of the valve. This paper describes the production of appropriate porosity in freeze-dried porcine pulmonary valve leaflets. We found that Tg' for this material is -83 degrees C, which made it impracticable to freeze-dry exclusively from the glassy state. Uncontrolled freeze-drying produced a variable structure with most of the pores considerably smaller than the desired size and a dense layer, apparently devoid of perforations, on the surface. Compacted layers also occurred within the substance of the leaflets. These appearances suggested that extensive collapse had occurred during the drying process. Variation of the cooling rate, the primary drying temperature, and the warming rate during secondary drying enabled us to identify the following conditions that provided satisfactory internal porosity: cooling at 5 degrees C/min, vacuum drying for 6 h at -20 degrees C, and secondary drying for 10 h during rewarming at 0.06-0.08 degrees C/min. The internal cavities measured 100-350 microns2 by ca. 400 microns2, which is adequate to provide access for the fibroblasts (cross-sectional area ca. 150-200 microns2 when rounded but fusiform when attached. However, the internal porous structure rarely communicated with the surface and mechanical perforation was required to provide continuity between the surface and the internal sponge. The resulting method provides a basis for studies of cell colonization.

Animals↗
PubMed1997

Fractures in cryopreserved elastic arteries.

The aim of this study was to define the conditions under which macroscopic fractures occur in vascular tissue during cryopreservation and to develop a practical cryopreservation method that prevents fracturing. The common carotid artery of the rabbit was subjected to a cryopreservation process that has been optimised for retention of in vitro function and cytological structure. This involves the stepwise addition and subsequent removal of dimethyl sulfoxide using a calculated protocol that avoids osmotic injury and minimises toxic action, controlled cooling, storage at -180 degrees C, and rapid warming. Seventy-five percent of such arteries were grossly fractured. The cooling and warming conditions were systematically varied to determine when in the cooling/storage/warming process the fractures occurred. Differential scanning calorimetry was then used to identify any corresponding thermal events. It was found that the fractures occurred as the temperature range -150 to -100 degrees C was traversed during the warming phase of the process. The glass transition temperature of a maximally freeze-concentrated solution of the cryoprotectant used was found to be -123 degrees C. Reducing the warming rate between the storage temperature (-180 degrees C) and -100 degrees C to < 50 degrees C/min prevented the fractures. Subsequent thawing could then be carried out rapidly in a 37 degrees C water bath without risk of fracture. We suggest that the fractures probably result from the thermal stresses created by rapid warming of the vitreous material that is produced by freeze-concentration of the aqueous phase. Relatively slow warming to -100 degrees C, at which temperature the vitreous material has softened, reduces these stresses and avoids the fractures.

Animals↗
PubMed1997

Repopulation of freeze-dried porcine valves with human fibroblasts and endothelial cells.

BACKGROUND AND AIMS OF THE STUDY: There is a need for a replacement cardiac valve constructed from non-immunogenic materials but incorporating living, and preferably autologous, cells. The object of this study was to colonize freeze-dried porcine valve leaflets with human fibroblasts and vascular endothelial cells. METHODS: Porcine pulmonary valve leaflets were freeze-dried to produce a porous matrix having communicating cavities of appropriate dimensions for fibroblast repopulation. Cultured human fibroblasts and vascular endothelial cells that had been cryopreserved by standard methods were added to freeze-dried leaflets. Following culture at 37 degrees C, the leaflets were examined by confocal scanning microscopy and transmission electron microscopy. RESULTS: Mechanical perforation of the leaflet surface permitted colonization of the freeze-dried matrix by fibroblasts; under the conditions we studied, the cell density did not reach physiologic levels but those cells that were present were well attached and metabolically active. Gentle cotton abrasion of the surface of the freeze-dried leaflets provided a suitable substrate for endothelial cell attachment and confluence was achieved in 10 days. Leaflets were perforated, cultured with human fibroblasts for 10 days, then gently rubbed with a cotton bud and cultured for a further 10 days with human endothelial cells. The endothelial cells formed a confluent layer on the surface and viable fibroblasts were present within the substance of the leaflet. CONCLUSION: Although these results are preliminary, they demonstrate the basic feasibility of this approach to the production of xenogeneic valves that contain the patient's own cells.

Animals↗
PubMed1997

Cryopreservation of rabbit corneas in dimethyl sulfoxide.

PURPOSE: To minimize the injury to endothelial cells during cryopreservation of rabbit corneas with dimethyl sulfoxide. METHODS: Rabbit corneas were cryopreserved using 20% wt/wt dimethyl sulfoxide (Me2SO), added and removed in stages to maintain the osmotically induced excursions in cell volume to within +/-40% of their isotonic volume. The vehicle solution, cooling rate, and conditions of storage used were those already reported to be optimal for endothelial cell survival after exposure to low temperatures. Survival was assessed by confocal microscopy with vital staining and by the ability of the endothelium to control stromal hydration during 3 hours of normothermic perfusion. The effect of temperature of addition and removal of Me2SO (room temperature [RT] or 2 degrees C) on endothelial viability also was measured. RESULTS: After thawing, all the cryopreserved corneas appeared structurally intact when assessed by vital staining and could limit stromal swelling during subsequent normothermic perfusion. Analysis of the rate of stromal swelling during the first 1.5 hours of normothermic perfusion indicated a substantial benefit when the Me2SO was removed at RT. Adding and removing the Me2SO at RT, which allowed a briefer exposure to Me2SO before cooling, resulted in better structural integrity of the endothelial layer than when the addition of cryoprotectant took place on ice. CONCLUSIONS: These results demonstrate the importance of osmotic stresses in the generation of injury to corneal endothelium during cryopreservation and the possibility of eventual successful cryopreservation of this tissue.

Animals↗
PubMed1996

The effect of cooling rate and temperature on the toxicity of ethylene glycol in the rabbit internal carotid artery.

The smooth muscle and vascular endothelium of small elastic arteries (the rabbit common carotid artery) are injured by exposure to 40% ethylene glycol (EG) at 4 degrees C, and additional damage occurs when the arteries are cooled without freezing to -20 degrees C. This paper reports attempts to reduce this injury by altering the cooling rate and temperature of exposure to the cryoprotectant. Very slow cooling (0.1 degree C/min) removed all residual smooth muscle and endothelial function when assessed in vitro after rewarming and removal of the cryoprotectant. Very rapid cooling to -20 degrees C also increased the injury, both to the endothelium and to the smooth muscle. Reducing the temperature of exposure to 40% EG from +4 degrees C to -20 degrees C had no beneficial effect on the smooth muscle but enabled the vascular endothelium to retain some functional activity. These data suggest that the mechanism responsible may be related to the physical properties of ethylene glycol rather than to a biochemical interaction with metabolic processes, and that it is a mechanism which is highly specific for the cell types involved. It also underlines the difficulties involved in the successful cryopreservation of complex tissues and organs.

Animals↗
PubMed1995

Cryopreservation of the common carotid artery of the rabbit: optimization of dimethyl sulfoxide concentration and cooling rate.

This paper describes the continuation of studies that demonstrated the suitability of CP-Tes solution as a medium for the introduction and removal of dimethyl sulfoxide in rabbit common carotid arteries and established the kinetics of cryoprotectant permeation in that tissue. In this paper we report the tolerance of rabbit common carotid artery to dimethyl sulfoxide, in concentrations up to 30% (w/w), using a technique of exposure that was designed to control osmotic stress. The maximum concentration achieved without damage was 15% (w/w). Vessels were then equilibrated with 15% dimethyl sulfoxide and cooled to -80 degrees C at 0.22, 0.69, 2.15, or 9.63 degrees C/min: they were then transferred to the gas phase of a liquid nitrogen refrigerator (temperature below -160 degrees C) for storage. Thawing was carried out in a 37 degrees C water bath. The optimum rate of cooling for these conditions was found to be 0.69 degrees C/min. The maximal recovery of contractile force in response to 10(-6) M norepinephrine was 30-40%; relaxation to acetylcholine (an endothelium-mediated function) was 80% of control, and an estimated 71% of endothelial cells survived with minimal ultrastructural change.

Acetylcholine↗
PubMed1994

Cryopreservation of the common carotid artery of the rabbit.

We describe experiments on the cryopreservation of the rabbit common carotid artery aimed at improving upon previous results. We describe the design of a double clamp which holds the artery during transportation and storage, preventing twisting, shortening, and collapse of the vessel. The device allowed perfusion with solutions as desired and markedly reduced the extent of endothelial loss during procurement and processing. We also studied the effects of three vehicle solutions; a modified Hanks' solution, a solution originally developed for the cryopreservation of smooth muscle (K-Pipes), and a solution designed for corneal endothelium (CP-Tes). The criteria used to make the assessments were smooth muscle contractility and the structure and function of the vascular endothelium. A new staining method for vascular endothelium (combining propidium iodide with silver nitrate) is described. We found that there was significantly more endothelial cell damage in rabbit carotid arteries frozen in Hanks' solution than in the other solutions, and the recovery of smooth muscle contractility was lowest in the Hanks' group. Arteries cryopreserved using CP-Tes as the vehicle solution showed less endothelial cell damage than arteries preserved with either K-Pipes or Hanks' solution, and these arteries also exhibited the greatest relaxation response to acetylcholine. We conclude that careful handling of the vessels is important; of the solutions studied, CP-Tes is preferred for the cryopreservation of rabbit carotid artery with Me2SO.

Animals↗
PubMed1994

Permeation of rabbit common carotid arteries with dimethyl sulfoxide.

Proton nuclear magnetic resonance has been used to measure the kinetics of permeation of dimethyl sulfoxide in the common carotid artery of the rabbit. The process is described by the following exponential equations, where t = time in minutes: % Unexchanged at 2 degrees C = 70e-0.515t + 30e-0.104t and %Unexchanged at 22 degrees C = 70e-1.790t + 30e-0.146t. The times required for 95% equilibration were 13 min at 22 degrees C and 18 min at 2 degrees C. The corresponding times for 99% equilibration were 24 and 32 min, respectively.

Animals↗
PubMed1994

Fractures in cryopreserved arteries.

The common carotid artery of the rabbit, a typical small elastic artery, can be cryopreserved using dimethyl sulfoxide, slow cooling, storage at less than -160 degrees C, and rapid warming. This technique provides satisfactory preservation of muscle and endothelial cells, but in about 75% of cases, gross circumferential fractures occur in the vessel wall. This paper investigates the influence of vehicle solution composition, cryoprotectant concentration, cooling rate, and storage temperature on the occurrence of cracks. When cooling was halted at -80 degrees C and the arteries were stored at this temperature, fractures no longer occurred. Possible mechanisms are discussed and it is proposed that mechanical stresses develop in the vitreous material that separates the ice crystals and lead to structural failure.

Animals↗
PubMed1993

The effect of polyvinylpyrrolidone and the cooling rate during corneal cryopreservation.

The effect upon endothelial cell survival of (a) PVP and (b) the cooling rate was investigated during the cryopreservation of rabbit corneas with 3 mol/liter dimethyl sulfoxide (Me2SO) dissolved in a hyperkalemic buffer vehicle solution that we designated CPTES; this solution was designed specifically to restrict deleterious ionic imbalances and cell swelling during hypothermic procedures. Polyvinylpyrrolidone (PVP) was used as the colloid and the corneas were cooled at 0.03, 0.1, 1, 25, or 125 degrees C/min, using the minimum amount of extracellular solution. Electron microscopy as well as staining with fluorescein diacetate and ethidium bromide (FDA/EB) was used to assess cellular integrity. To reduce osmotic stress, steps for the serial equilibration of the cryoprotectant additives (CPAs) were based upon calculations that predict endothelial volume during CPA exchange. A toxicity study showed that at 0 degrees C all the CPA equilibration protocols were well tolerated; for example, FDA/EB staining indicated that 97% intact cells were retained following direct transfer to 3 mol/liter Me2SO in CPTES to which 40% w/v PVP had been added as an osmotic buffer. However, less than 20% of cells were intact by FDA/EB staining with all corneas frozen at rates > 1 degree C/min regardless of which equilibration protocol was employed, nor were there any intact cells when 3 mol/liter Me2SO in CPTES was used alone at the lowest cooling rate. At intermediate cooling rates viability was improved: the highest mean survival of 81% was obtained using 3 mol/liter Me2SO in CPTES plus 40% PVP. Electron microscopy showed that detachment of the endothelial layer often occurred, but least damage was evident following exposure to 3 mol/liter Me2SO in CPTES plus 40% PVP and cooling at 1 degree C/min. No thawed cornea could maintain normal control of hydration immediately upon return to isotonic medium. The results show that, with these cryopreservation protocols, loss of cell integrity occurs at cooling rates greater than 1 degree C/min, whereas at lower rates higher survival of individual cells was achieved, but cellular adhesion to the basement membrane was impaired.

Animals↗
PubMed1993

The possibility of resuscitating livers after warm ischemic injury.

The number of clinical liver transplants that can be performed is limited by the availability of suitable donor organs. If it were possible to harvest and use livers after cardiac arrest, the supply could be improved. The mechanisms of damage in warm ischemia are not yet well understood and the consequences of transplanting a liver that is unable to provide immediate life-support are unacceptable. This study aims to identify areas for more detailed study in an attempt to improve the quality of livers harvested after significant warm ischemia, and to select acceptable organs for transplantation. Porcine livers were subjected to 75 min of warm ischemia and then perfused at 37 degrees C for 3 hr, during which period biochemical monitoring was carried out. At the end of the perfusion, histological and transmission electron microscopical studies were made. Large amounts of the intracellular enzymes ALT, AST, and LDH were released into the perfusate during the first 30 min of perfusion, but this--and the further amounts released during the subsequent 2.5 hr--was influenced by the composition of the perfusate. The inclusion of the substrates fructose and oleate, plus amino acids, substantially reduced this release and also improved the ability of the livers to metabolize ammonia. Oxygen free-radical scavengers had a significant, but smaller, beneficial effect. Electron microscopy confirmed the value of perfusion in improving cell morphology, and the additional value of including metabolic substrates. This study shows that hepatocellular structure and function can be improved by appropriate perfusion methods that also provide a simple means of monitoring some important functions. Both metabolic support and neutralization of oxygen free-radical action have a role to play in this approach to rendering ischemically injured livers acceptable for clinical use.

Alanine Transaminase↗
PubMed1992

MRC funding.

Explore the source record for details and available documents.

Financing, Government↗
PubMed1992

Experimental results on the rewarming of a cryopreserved organ phantom in a UHF field.

We describe a UHF rewarming system which has been used to measure warming rates, and particularly the uniformity of warming on three orthogonal axes, in a rabbit kidney phantom 36 mm in diameter. The stabilizing effect is demonstrated of using an E-field directed along the temperature gradient (or normal to any surface of dielectric discontinuity). One hot spot remains unaccounted for. The average warming rates and power dissipation are related to the volumetric heat capacity of the phantom material at -30 degrees C.

Animals↗