Influence of dermal equivalent maturation on the development of a cultured skin equivalent.

Histologic and immunofluorescence methods were used to analyse the presence of fibronectin, chondroitin-4-sulphate and chondroitin-6-sulphate, type III and IV collagens, laminin, and keratins to assess the maturation level of cultured dermal and skin equivalents. In a first phase, fibroblasts in monolayer culture were compared with dermal equivalents in which fibroblasts are embedded in a type I collagen gel. Different fluorescent patterns were observed depending on the culture system used. A sequential appearance of macromolecules was noticed in dermal equivalents. Fibronectin was first detected after 4 days of culture, whereas chondroitin-4-sulphate and chondroitin-6-sulphate and type III collagen were present after 7 days. In contrast, all three macromolecules were detected at 24 h of culture in fibroblastic monolayer cultures. In a second phase, the quality of our skin equivalents was evaluated according to the seeding time of epidermal cells upon dermal equivalents (1, 4, or 7 days). A satisfactory stratification was obtained when keratinocytes were seeded after 4 and 7 days of dermal equivalent culture. Laminin and fibronectin were detected at the dermo-epidermal junction, but type IV collagen was absent. Various keratins, as detected by the AE1, AE2, and AE3 antibodies, were present in the epidermal layer. Following keratinocyte confluence, a change in the organization pattern of type III collagen in the dermal fraction of the skin equivalent was also noticed. Our comparative results show that seeding of epidermal cells on a more mature dermal equivalent leads to improved differentiation status of the epidermal layer.     

The evaluation of the neutron dose equivalent in the two-bend maze

The purpose of this study was to explore the effect of the second bend of the maze, on the neutron dose equivalent, in the 15 MV linear accelerator vault, with two bend maze. These two bends of the maze were covered by 32 points where the neutron dose equivalent was measured. There is one available method for estimation of the neutron dose equivalent at the entrance door of the two bend maze which was tested using the results of the measurements. The results of this study show that the neutron equivalent dose at the door of the two bend maze was reduced almost three orders of magnitude. The measured TVD in the first bend (closer to the inner maze entrance) is about 5 m. The measured TVD result is close to the TVD values usually used in the proposed models for estimation of neutron dose equivalent at the entrance door of the single bend maze. The results also determined that the TVD in the second bend (next to the maze entrance door) is significantly lower than the TVD values found in the first maze bend.     

Study of microdosimetric energy deposition patterns in tissue-equivalent medium due to low-energy neutron fields using a graphite-walled proportional counter

To improve radiation protection dosimetry for low-energy neutron fields encountered in nuclear power reactor environments, there is increasing interest in modeling neutron energy deposition in metrological instruments such as tissue-equivalent proportional counters (TEPCs). Along with these computational developments, there is also a need for experimental data with which to benchmark and test the results obtained from the modeling methods developed. The experimental work described in this paper is a study of the energy deposition in tissue-equivalent (TE) medium using an in-house built graphite-walled proportional counter (GPC) filled with TE gas. The GPC is a simple model of a standard TEPC because the response of the counter at these energies is almost entirely due to the neutron interactions in the sensitive volume of the counter. Energy deposition in tissue spheres of diameter 1, 2, 4 and 8 μm was measured in low-energy neutron fields below 500 keV. We have observed a continuously increasing trend in microdosimetric averages with an increase in neutron energy. The values of these averages decrease as we increase the simulated diameter at a given neutron energy. A similar trend for these microdosimetric averages has been observed for standard TEPCs and the Rossi-type, TE, spherical wall-less counter filled with propane-based TE gas in the same energy range. This implies that at the microdosimetric level, in the neutron energy range we employed in this study, the pattern of average energy deposited by starter and insider proton recoil events in the gas is similar to those generated cumulatively by crosser and stopper events originating from the counter wall plus starter and insider recoil events originating in the sensitive volume of a TEPC.     

Folding-unfolding energy change of a simple sphere model protein and an energy landscape of the folding process

We have calculated the free energy of a spherical model of a protein or part of a protein generated in the way of protein folding. Two spherical models are examined; one is a homogeneous model consisting of only one residue type—hydrophobic. The other is a heterogeneous model consisting of two residue types—strong hydrophobic and weak hydrophobic. Both models show a folding transition state, and the latter model reproduces the trend of the experimental folded-unfolded energy change. The heterogeneous model suggests that in the folding process of a protein of more than 70 residues, a specific region of the protein folds first to form a stable region, then the other residues follow the folding process. The energy landscape of folding of a small protein is approximately a funnel model, whereas a flatter energy landscape is suggested for larger proteins of more than 55–70 residues. Proteins 33:408–416, 1998. © 1998 Wiley-Liss, Inc.     

Water transport and IIF parameters for a connective tissue equivalent

Understanding the biophysical processes that govern freezing injury of a tissue equivalent (TE) is an important step in characterizing and improving the cryopreservation of these systems. TEs were formed by entrapping human dermal fibroblasts (HDFs) in collagen or in fibrin gels. Freezing studies were conducted using a Linkam cryostage fitted to an optical microscope allowing observation of the TEs cooled under controlled rates between 5 and 130 degrees C/min. Typically, freezing of cellular systems results in two biophysical processes that are both dependent on the cooling rate: dehydration and/or intracellular ice formation (IIF). Both these processes can potentially be destructive to cells. In this study, the biophysics of freezing cells in collagen and fibrin TEs have been quantified and compared to freezing cells in suspension. Experimental data were fitted in numerical models to extract parameters that governed water permeability, E(Lp) and L(pg), and intracellular ice nucleation, omega(o) and kappa(o). Results indicate that major differences exist between freezing HDFs in suspension and in a tissue equivalent. During freezing, 55% of the HDFs in suspension formed IIF as compared to 100% of HDFs forming IIF in collagen and fibrin TE at a cooling rate of 130 degrees C/min. Also, both the water permeability and the IIF parameters were determined to be higher for HDFs in TEs as compared to cell suspensions. Between the TEs, HDFs in fibrin TE exhibited higher values for the biophysical parameters as compared to HDFs in collagen TE. The observed biophysics seems to indicate that cell-cell and cell-matrix interactions play a major role in ice propagation in TEs.     

Genetic influences on tissue deposition of aluminum in mice

Aluminum is a known neurotoxin and has been suggested to play a role in the development of Senile Dementia of the Alzheimer's Type. The relationship between aluminum exposure and senile dementia cannot be a simple one, however, as not all exposure results in neurotoxic manifestations. To determine if there are genetic differences in susceptibility to moderate aluminum exposure, 16 mice of five inbred strains were divided into two groups. The control group was fed a purified diet containing all known requirements for mice; the experimental group was fed the same diet supplemented with 260 mg Al/kg diet for 28 d. Analysis of brains, livers, and tibias for aluminum concentrations revealed strain differences in response to dietary treatment. The most notable results occurred in the DBA/2 and C3H/2 strains, with brain aluminum levels higher in the experimental groups. In contrast, A/J, BALB/c, and C57BL/6 strains showed no differences in brain aluminum in response to dietary treatment. These findings suggest that there are genetic differences in the permeability of the blood brain barrier and lend support to the hypothesis that variability in aluminum toxicity may be, in part, genetically determined.     

Acceptance of a connective tissue equivalent for grafting and capsuloligamentary reconstruction

A strip of a connective tissue equivalent prepared by using the patient's fibroblasts cultivated in vitro and calf skin collagen, was used for capsuloligamentary reconstruction of the knee. The study of the humoral and cell-mediated immune responses indicated that there was no cell-mediated immune reaction and only a transient humoral reaction 2 months after implantation. This first assay in human surgery gave a good functional result, and an immunological response was no longer observed 5 months after grafting.     

Monte Carlo simulation of neutron dose equivalent by photoneutron production inside the primary barriers of a radiotherapy vault

PurposeTo evaluate the neutron dose equivalent produced by photoneutrons inside the primary barriers of a radiotherapy vault.MethodsMonte Carlo simulations were performed for investigating the production of photoneutrons as well as neutron shielding requirements. Two photon beams of 15 and 18 MV struck sheets of steel and lead, and the neutron doses were calculated at the isocenter (P_iso) and at a distance of 50 cm from the inside wall (P_wall) while delivering 1 Gy to the patient. The proper thicknesses of borated polyethylene (BPE) and concrete were simulated to reduce neutron contamination.ResultsWhen the primary barrier consisted of a concrete alone, the neutron doses at P_iso were 0.5 μSv/Gy and 12.8 μSv/Gy for 15- and 18-MV, respectively. At P_wall, the neutron doses were 15.8 μSv/Gy and 318.4 μSv/Gy for 15- and 18-MV, respectively. When 15 MV photons interacted with metal sheets, the neutron doses were 0.4–22.2 μSv/Gy at P_iso and 15.8–812.5 μSv/Gy at P_wall, depending on the thickness and material of the metal sheets and neutron shielding. In the case of 18 MV photons with the same configuration, the neutron doses were 0.9–59.5 μSv/Gy and 73.9–5006.1 μSv/Gy for P_iso and P_wall, respectively. The neutron dose delivered to the patient was reduced to the level of the dose delivered with a concrete barrier by including a 10-cm-thick BPE for each beam.ConclusionsWhen the primary barrier shielding is designed with a metal sheet inside for high energy, proper neutron shielding should be constructed to avoid undesirable photoneutron dose.     

Determination of equivalent amounts of kinetic energy, work, and heat energy in the human body

The goal of this study is determine the mechanical equivalent of heat and the functional capacity of metabolism of walking at a slow pace (velocity = 4022m/hour, length of a step=75cm, energy utilization of a 70 kg person is 200kcal/hour). 50 healthy physicians were chosen randomly, and up and down motion of the body were determined as 6cm while stepping. Based on these, the heat equivalent is 37.5kcal/hour for horizontal motion and 52.7kcal/hour for 6cm up-and-down bobbing motions of body, and the functional capacity of metabolism is at least 45% ([37.5+52.7]/200=45%) for slow walking state, that this capacity is twofold more than earlier information. Muscle converts kinetic energy (work) to heat via friction, and heat sources of the body, and the concepts of thermogenesis and the functional capacity of metabolism should be revised.     

Measurements of neutron energy using a recoil-proton telescope and a high-pressure ionization chamber

Two very different techniques for measuring the energy of neutrons in the energy range 0.1-10 MeV are presented and compared. A recoil-proton spectrometer is used to determine the energy spectra of neutrons produced by the d(4)-Be and p(4)-Be reactions down to the low-energy threshold of 0.7 MeV. The same radiation fields are also measured with a recently developed method using a high-pressure ionization chamber that can be used to determine the mean energy of the neutrons in a mixed neutron-gamma radiation field provided the gamma-ray absorbed dose fraction is determined independently. An intercomparison of the two methods shows that the high-pressure ionization chamber compares well and supplements the established recoil-proton spectrometer technique. The almost isotropic response of the chamber has enabled measurements to be made of the variation of mean neutron energy with depth in water for the two radiation fields.     

Influence of dietary energy intake and substrate addition on the in vitro metabolism of glucose and glutamine in rumen epithelial tissue

Ten Holstein steers were fed either 14.2 or 26.2 Mcal ME for 28 days prior to investigating the effect of dietary energy on epithelial metabolism. Rumen papillae were incubated in vitro with glucose (5 mM) or glutamine (1 mM) as well as additional energy substrates. Increased dietary intake increased production of 14CO2 from glucose and glutamine, increased uptake and net lactate production from glucose, and decreased net glutamate and alanine production from glutamine. At these substrate concentrations, rates of glucose oxidation to 14CO2 were sevenfold higher than glutamine.     

Influence of mitochondrial creatine kinase on the mitochondrial/extramitochondrial distribution of high energy phosphates in muscle tissue: evidence for a leak in the creatine shuttle

The influence of mitochondrial creatine kinase on subcellular high energy systems has been investigated using isolated rat heart mitochondria, mitoplasts and intact heart and skeletal muscle tissue.In isolated mitochondria, the creatine kinase is functionally coupled to oxidative phosphorylation at active respiratory chain, so that it catalyses the formation of creatine phosphate against its thermodynamic equilibrium. Therefore the mass action ratio is shifted from the equilibrium ratio to lower values. At inhibited respiration, it is close to the equilibrium value, irrespective of the mechanism of the inhibition. The same results were obtained for mitoplasts under conditions where the mitochondrial creatine kinase is still associated with the inner membrane.In intact tissue increasing amounts of creatine phosphate are found in the mitochondrial compartment when respiration and/or muscle work are increased. It is suggested that at high rates of oxidative phosphorylation creatine phosphate is accumulated in the intermembrane space due to the high activity of mitochondrial creatine kinase and the restricted permeability of reactants into the extramitochondrial space. A certain amount of this creatine phosphate leaks into the mitochondrial matrix.This leak is confirmed in isolated rat heart mitochondria where creatine phosphate is taken up when it is generated by the mitochondrial creatine kinase reaction. At inhibited creatine kinase, external creatine phosphate is not taken up. Likewise, mitoplasts only take up creatine phosphate when creatine kinase is still associated with the inner membrane. Both findings indicate that uptake is dependent on the functional active creatine kinase coupled to oxidative phosphorylation.Creatine phosphate uptake into mitochondria is inhibited with carboxyatractyloside. This suggests a possible role of the mitochondrial adenine nucleotide translocase in creatine phosphate uptake.Taken together, our findings are in agreement with the proposal that creatine kinase operates in the intermembrane space as a functional unit with the adenine nucleotide translocase in the inner membrane for optimal transfer of energy from the electron transport chain to extramitochondrial ATP-consuming reactions.     

Collagen deposition in the subcutaneous tissue during wound healing in humans: a model evaluation

Wound healing encompasses coagulation, inflammation, angiogenesis, fibroplasia, contraction, epithelialisation and remodeling. A granulation tissue is produced following incision of tissue such as skin, abdominal wall or the gastrointestinal tract, and the strength of the wound is determined primarily by the collagen content early in the healing course. Few models are available to study wound healing in man. The percutaneous insertion of expanded poly-tetrafluoroethylene tubes (ePTFE) into the subcutaneous tissue has been an established model for 20 years. The procedure is performed using a local anesthesia. The model has a diameter of 2.5 mm, a length of 5-10 cm and a pore size of 90-120 microns which is substantially more than that of vascular grafts. The polymer accumulates granulation tissue, the architecture of which resembles that of a normal surgical wound. Previous studies on the use of the ePTFE model in wound healing research are summarized in detail. Histological and immunohistochemical analyses of the granulation tissue deposited in the model were undertaken. The content of amino acids following hydrolysis of the granulation tissue was determined applying spectrophotometric or HPLC assays. Collagen amounts accumulated in the model are expressed as hydroxyproline per length of ePTFE or per total protein. Following a study in rats we examined 85 healthy volunteers and 158 surgical patients in the studies. Higher contents of hydroxyproline were found 10 days after implantation as compared to 5 days with considerable inter-person variation. Regarding median values there was a 25% difference between two measurements performed on two distinct ePTFE tubes from the same person, and a 12% difference between values obtained from two different pieces of the same ePTFE. Higher accumulation levels of hydroxyproline did not result in higher variability. Deposition of proline in the model correlated closely to total protein content. The ePTFE and a modified PVA model were compared in surgical patients. No reproducible measurements of hydroxyproline deposition were obtained with the PVA model as opposed to the ePTFE model. It is concluded that the modified PVA model is inadequate for determination of collagen deposition in subcutaneous granulation tissue. We found no correlation between collagen deposition levels obtained with placement of the ePTFE model in the subcutaneous tissue of the arm and in an uncomplicated surgical wound of the groin in the same patient, respectively. Significantly higher collagen deposition levels in the model were found in the surgical wound. Conversely, there was a significant correlation between protein deposition levels obtained at the two sites. Patients undergoing minor surgery (groin hernia repair) did not differ from healthy non-traumatized volunteers as regards deposition of collagen in subcutaneous tissue of the arm, whereas patients subjected to major general surgery demonstrated a significant decline during the postoperative phase compared to a preoperative evaluation. This decline was enhanced in patients who had infectious complications. Non-smoking volunteers were found to specifically accumulate more collagen (median value 82%) than smokers matched for age and gender. Irrespective of the smoking status women accumulated significantly more collagen in the model than men. These findings were re-tested in a prospective series leading to the same conclusion. Matrix metalloproteinases (MMP-2 and MMP-9) were determined in wound fluid obtained from the subcutaneous cavities of herniotomy wounds 24 and 48 h after operation. A significant and inverse correlation was demonstrated between MMP-9 after 24 h and accumulation levels of collagen in the ePTFE tube 10 days after implantation in the wound. Finally, it was demonstrated that local application of granulocyte-macrophage colony-stimulating factor into the ePTFE model during implantation specifically and dose-dependently reduced the number of fibroblasts and deposition of collagen. The doses chosen for the experiments resulted in both a local and a systemic effect. It is concluded that the minimally invasive ePTFE model, despite a certain level of variability, presently provides one of the best possibilities of evaluation of the wound healing potential in both volunteers and patients under various conditions. We found the model convenient for the assessment of both matrix deposition during wound healing and the influence of several factors including demographic characteristics, trauma, tobacco smoking, drugs and tissue degrading components of the wound.     

The use of human fibroblasts grown on microcarriers for the formation of the connective tissue equivalent

Live equivalents of tissues, specifically those produced on the basis of fibroblasts and collagen gel, are widely used for repair of organ and tissues defects. In clinical practice, it is more convenient to use the fibroblasts grown on microcarriers or such a connective tissue equivalent when the fibroblasts on microcarriers are embedded in collagen gel. We studied the properties of a connective tissue equivalent produced by embedding the fibroblasts grown on microcarriers in collagen gel for its prospective use in clinical practice. According to our results, the optimal time of use of the live tissue equivalent amounts to three--four days after embedding of fibroblasts on microcarriers in gel. At that time, contraction only begins, which facilitates manipulations with the gel.