Gas exchange is essential for bioreactor cultivation of tissue engineered cartilage

Tissue engineered cartilage can be grown in vitro if the necessary physical and biochemical factors are present in the tissue culture environment. Cell metabolism and tissue composition were studied for engineered cartilage cultured for 5 weeks using bovine articular chondrocytes, polymer scaffolds (5 mm diameter x 2 mm thick fibrous discs), and rotating bioreactors. Medium pH and concentrations of oxygen, carbon dioxide, glucose, lactate, ammonia, and glycosoaminoglycan (GAG) were varied by altering the exchange rates of gas and medium in the bioreactors. Cell-polymer constructs were assessed with respect to histomorphology, biochemical composition and metabolic activity. Low oxygen tension ( approximately 40 mmHg) and low pH ( approximately 6.7) were associated with anaerobic cell metabolism (yield of lactate on glucose, YL/G, of 2.2 mol/mol) while higher oxygen tension ( approximately 80 mmHg) and higher pH ( approximately 7.0) were associated with more aerobic cell metabolism (YL/G of 1.65-1.79 mol/mol). Under conditions of infrequent medium replacement (50% once per week), cells utilized more economical pathways such that glucose consumption and lactate production both decreased, cell metabolism remained relatively aerobic (YL/G of 1.67 mol/mol) and the resulting constructs were cartilaginous. More aerobic conditions generally resulted in larger constructs containing higher amounts of cartilaginous tissue components, while anaerobic conditions suppressed chondrogenesis in 3D tissue constructs.     

Oxygen gradients in tissue-engineered PEGT/PBT cartilaginous constructs: measurement and modeling

The supply of oxygen within three-dimensional tissue-engineered (TE) cartilage polymer constructs is mainly by diffusion. Oxygen consumption by cells results in gradients in the oxygen concentration. The aims of this study were, firstly, to identify the gradients within TE cartilage polymer constructs and, secondly, to predict the profiles during in vitro culture. A glass microelectrode system was adapted and used to penetrate cartilage and TE cartilaginous constructs, yielding reproducible measurements with high spatial resolution. Cartilage polymer constructs were cultured for up to 41 days in vitro. Oxygen concentrations, as low as 2-5%, were measured within the center of these constructs. At the beginning of in vitro culture, the oxygen gradients were steeper in TE constructs in comparison to native tissue. Nevertheless, during the course of culture, oxygen concentrations approached the values measured in native tissue. A mathematical model was developed which yields oxygen profiles within cartilage explants and TE constructs. Model input parameters were assessed, including the diffusion coefficient of cartilage (2.2 x 10(-9)) + (0.4 x 10(-9) m(2) s(-1)), 70% of the diffusion coefficient of water and the diffusion coefficient of constructs (3.8 x 10(-10) m(2) s(-1)). The model confirmed that chondrocytes in polymer constructs cultured for 27 days have low oxygen requirements (0.8 x 10(-19) mol m(-3) s(-1)), even lower than chondrocytes in native cartilage. The ability to measure and predict local oxygen tensions offers new opportunities to obtain more insight in the relation between oxygen tension and chondrogenesis.     

The effect of oxygen tension on human articular chondrocyte matrix synthesis: Integration of experimental and computational approaches

Significant oxygen gradients occur within tissue engineered cartilaginous constructs. Although oxygen tension is an important limiting parameter in the development of new cartilage matrix, its precise role in matrix formation by chondrocytes remains controversial, primarily due to discrepancies in the experimental setup applied in different studies. In this study, the specific effects of oxygen tension on the synthesis of cartilaginous matrix by human articular chondrocytes were studied using a combined experimental‐computational approach in a “scaffold‐free” 3D pellet culture model. Key parameters including cellular oxygen uptake rate were determined experimentally and used in conjunction with a mathematical model to estimate oxygen tension profiles in 21‐day cartilaginous pellets. A threshold oxygen tension (pO₂ ≈ 8% atmospheric pressure) for human articular chondrocytes was estimated from these inferred oxygen profiles and histological analysis of pellet sections. Human articular chondrocytes that experienced oxygen tension below this threshold demonstrated enhanced proteoglycan deposition. Conversely, oxygen tension higher than the threshold favored collagen synthesis. This study has demonstrated a close relationship between oxygen tension and matrix synthesis by human articular chondrocytes in a “scaffold‐free” 3D pellet culture model, providing valuable insight into the understanding and optimization of cartilage bioengineering approaches. Biotechnol. Bioeng. 2014;111: 1876–1885. © 2014 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Control of oxygen uptake, microcirculation and glucose release by circulating noradrenaline in perfused rat liver

The effect of noradrenaline on oxygen uptake, on periportal and perivenous oxygen tension at surface acini, on microcirculation and on glucose output were studied in isolated rat livers perfused at constant flow with Krebs-Henseleit-hydrogen carbonate buffer containing 5mM glucose and 2mM lactate. Noradrenaline at 1 microM concentration caused a decrease in oxygen uptake, while at 0.1 microM it led to an increase. Both high and low doses of noradrenaline decreased the tissue surface oxygen tension in periportal and - after a transient rise - in perivenous areas. Noradrenaline at an overall constant flow caused an increase of portal pressure and an alteration of the intrahepatic distribution of the perfusate: at the surface of the liver and in cross sections infused trypan blue led to only a slightly heterogeneous staining after a low dose of noradrenaline but to a clearly heterogeneous staining after a high dose. Both high and low doses of noradrenaline stimulated glucose release. All effects could be inhibited by the alpha-blocking agent phentolamine. In conclusion, control of hepatic oxygen consumption by circulating noradrenaline is a complex result of opposing hemodynamic and metabolic components: the microcirculatory changes inhibit oxygen uptake; they dominate after high catecholamine doses. The metabolic effects include a stimulation of oxygen utilization; they prevail at low catecholamine levels. The noradrenergic control of glucose release is also very complex, involving direct, metabolic and indirect, hemodynamic components.     

Cartilage Tissue Engineering: Controversy in the Effect of Oxygen

Articular cartilage lacks the ability to repair itself and consequently defects in this tissue do not heal. Tissue engineering approaches, employing a scaffold material and cartilage producing cells (chondrocytes), hold promise for the treatment of such defects. In these strategies the limitation of nutrients, such as oxygen, during in vitro culture are of major concern and will have implications for proper bioreactor design. We recently demonstrated that oxygen gradients are indeed present within tissue engineered cartilaginous constructs. Interestingly, oxygen, besides being an essential nutrient, is also a controlling agent of developmental processes including cartilage formation. However, the specific role of oxygen in these processes is still obscure despite the recent advances in the field. In particular, the outcome of published investigations is inconsistent regarding the effect of oxygen tension on chondrocytes. Therefore, this article describes the possible roles of oxygen gradients during embryonic cartilage development and reviews the data reported on the effect of oxygen tension on in vitro chondrocyte proliferation and differentiation from a tissue engineering perspective. Furthermore, possible causes for the variance in the data are discussed. Finally, recommendations are included that may reduce the variation, resulting in more reliable and comparable data.     

Concentric cylinder bioreactor for production of tissue engineered cartilage: effect of seeding density and hydrodynamic loading on construct development

A concentric cylinder bioreactor has been developed to culture tissue engineered cartilage constructs under hydrodynamic loading. This bioreactor operates in a low shear stress environment, has a large growth area for construct production, allows for dynamic seeding of constructs, and provides for a uniform loading environment. Porous poly-lactic acid constructs, seeded dynamically in the bioreactor using isolated bovine chondrocytes, were cultured for 4 weeks at three seeding densities (60, 80, 100 x 10(6) cells per bioreactor) and three different shear stresses (imposed at 19, 38, and 76 rpm) to characterize the effect of chondrocyte density and hydrodynamic loading on construct growth. Construct seeding efficiency with chondrocytes is greater than 95% within 24 h. Extensive chondrocyte proliferation and matrix deposition are achieved so that after 28 days in culture, constructs from bioreactors seeded at the highest cell densities contain up to 15 x 10(6) cells, 2 mg GAG, and 3.5 mg collagen per construct and exhibit morphology similar to that of native cartilage. Bioreactors seeded with 60 million chondrocytes do not exhibit robust proliferation or matrix deposition and do not achieve morphology similar to that of native cartilage. In cultures under different steady hydrodynamic loading, the data demonstrate that higher shear stress suppresses matrix GAG deposition and encourages collagen incorporation. In contrast, under dynamic hydrodynamic loading conditions, cartilage constructs exhibit robust matrix collagen and GAG deposition. The data demonstrate that the concentric cylinder bioreactor provides a favorable hydrodynamic environment for cartilage construct growth and differentiation. Notably, construct matrix accumulation can be manipulated by hydrodynamic loading. This bioreactor is useful for fundamental studies of construct growth and to assess the significance of cell density, nutrients, and hydrodynamic loading on cartilage development. In addition, studies of cartilage tissue engineering in the well-characterized, uniform environment of the concentric cylinder bioreactor will develop important knowledge of bioprocessing parameters critical for large-scale production of engineered tissues.     

Both superficial and deep zone articular chondrocyte subpopulations exhibit the crabtree effect but have different basal oxygen consumption rates

In the absence of in vivo measurements, the oxygen concentration within articular cartilage is calculated from the balance between cellular oxygen consumption and mass transfer. Current estimates of the oxygen tension within articular cartilage are based on oxygen consumption data from full‐depth tissue samples. However, superficial and deep cell subpopulations of articular cartilage express intrinsic metabolic differences. We test the hypothesis that the subpopulations differ with respect to their intrinsic oxygen consumption rate. Chondrocytes from the full cartilage thickness demonstrate enhanced oxygen consumption when deprived of glucose, consistent with the Crabtree phenomena. Chondrocyte subpopulations differ in the prevailing availability of oxygen and glucose, which decrease with distance from the cartilage–synovial fluid interface. Thus, we tested the hypothesis that the oxygen consumption of each subpopulation is modulated by nutrient availability, by examining the expression of the Crabtree effect. The deep cells had a greater oxygen consumption than the superficial cells (V_max of 6.6 compared to 3.2 fmol/cell/h), consistent with our observations of mitochondrial volume (mean values 52.0 vs. 36.4 µm³/cell). Both populations expressed the Crabtree phenomena, with oxygen consumption increasing ～2.5‐fold in response to glycolytic inhibition by glucose deprivation or 2‐deoxyglucose. Over 90% of this increase was oligomycin‐sensitive and thus accounted for by oxidative phosphorylation. The data contributes towards our understanding of chondrocyte energy metabolism and provides information valuable for the accurate calculation of the oxygen concentration that the cells experience in vivo. The work has further application to the optimisation of bioreactor design and engineered tissues. J. Cell. Physiol. 223:630–639, 2010. © 2010 Wiley‐Liss, Inc.     

Oxygen consumption of equine articular chondrocytes: Influence of applied oxygen tension and glucose concentration during culture

We investigated the oxygen (O(2)) uptake of equine articular chondrocytes to assess their reactions to anoxia/re-oxygenation. They were cultured under 5% or 21% gas phase O(2) and at glucose concentrations of 0, 1.0 or 4.5g/L in the culture medium (n=3). Afterwards, the O(2) consumption rate of the chondrocytes was monitored (oxymetry) before and after an anoxia period of 25min. The glucose consumption and lactate release were measured at the end of the re-oxygenation period. The chondrocytes showed a minimal O(2) consumption rate, which was hardly changed by anoxia. Independently from the O(2) tension, glucose uptake by the cells was about 30% of the available culture medium glucose, thus higher for cells at 4.5g/L glucose (n=3). Lactate release was also independent from O(2) tension, but lower for cells at 4.5g/L glucose (n=3). Our observations indicated that O(2) consumption by equine chondrocytes was very low despite a functional mitochondrial respiratory chain, and nearly insensitive to anoxia/re-oxygenation. But the chondrocytes metabolism was modified by an excess of O(2) and glucose.     

Insulin secretion by rat lacrimal glands: effects of systemic and local variables

To understand the secretory mechanisms and physiological role of insulin in the tear film, the present study examined 1) the time course of insulin secretion in the tear film under glucose intravenous stimulation, 2) the glucose- and carbachol-induced insulin secretion from isolated lacrimal gland (LG), 3) the effect of insulin on glucose consumption by the cornea, and 4) the expression of insulin, pancreatic duodenal homeobox-1 (PDX-1), and glucose transport proteins (GLUTs) in LG tissue. The insulin level in the tear film of 8-wk-old male Wistar rats increased from 0.6 +/- 0.45 to 3.7 +/- 1.3 ng/ml in the initial minutes after glucose stimulation. In vitro assays demonstrated that higher glucose concentrations from 2.8 to 16.7 mM, 200 microM carbachol, or 40 mM KCl significantly increased insulin secretion from lacrimal glands compared with controls, but did not detect C-peptide as measured by RIA. Glucose consumption by corneal tissue, evaluated by radiolabeled D-[U-14C]glucose uptake, was 24.07 +/- 0.61 and was enhanced to 31.63 +/- 3.15 nmol x cornea(-1) x 2 h(-1) in the presence of 6 nM insulin (P = 0.033) and to 37.5 +/- 3.7 nmol x cornea(-1) x 2 h(-1) in the presence of 11.2 mM glucose (P = 0.015). Insulin and PDX-1 mRNA was detected in LG. Insulin was located in the apical areas of acinar cells by immunoperoxidase and the expression of GLUT-1, but not PDX-1, was confirmed by Western blot. These findings suggest that insulin secretion in the tear film is influenced by local stimuli such as nutrient and neural inputs and that this hormone plays a metabolic role in ocular surface tissues. These data also indicate that under normal conditions the insulin secreted by LG is stored, but it is not clear that is locally produced in the LG.     

Muscarinic receptors in pancreatic islets of the rat. Demonstration and dependence on long-term glucose environment

The presence of muscarinic receptors in islets of Langerhans was assessed by measurement of specific binding of [3H]methylscopolamine. Specific binding was defined as total binding minus binding obtained in the presence of 1000-fold or higher excess of unlabeled methylscopolamine. At 37 degrees C specific binding was significant after 1 min and plateaued after 10 min of incubation. Displacement of label by increasing concentrations of unlabeled methylscopolamine indicated a dissociation constant of 1.5 x 10(-12) M. Effects of methylscopolamine on insulin release were evaluated from the inhibitions of cholinergic-induced insulin release. 4 x 10(-10) M methylscopolamine inhibited acetylcholine (20 microM)-induced insulin release more than 60%. Binding was not influenced by the following variations during binding incubations: changing the glucose concentration from 0 to 8.3 mM, adding rotenon (1 microM) or omitting calcium from the incubation medium. Islets kept in tissue culture exhibited higher binding when cultured at 11.1 than at 3.3 mM glucose for 96 h. It is concluded that islets contain muscarinic receptors, the binding to which can be subject to alteration by the long-term glucose environment.     

Synoviocyte Derived-Extracellular Matrix Enhances Human Articular Chondrocyte Proliferation and Maintains Re-Differentiation Capacity at Both Low and Atmospheric Oxygen Tensions

BackgroundCurrent tissue engineering methods are insufficient for total joint resurfacing, and chondrocytes undergo de-differentiation when expanded on tissue culture plastic. De-differentiated chondrocytes show poor re-differentiation in culture, giving reduced glycosaminoglycan (GAG) and collagen matrix accumulation. To address this, porcine synoviocyte-derived extracellular matrix and low (5%) oxygen tension were assessed for their ability to enhance human articular chondrocyte expansion and maintain re-differentiation potential.MethodsPorcine synoviocyte matrices were devitalized using 3 non-detergent methods. These devitalized synoviocyte matrices were compared against tissue culture plastic for their ability to support human chondrocyte expansion. Expansion was further compared at both low (5%), and atmospheric (20%) oxygen tension on all surfaces. Expanded cells then underwent chondrogenic re-differentiation in aggregate culture at both low and atmospheric oxygen tension. Aggregates were assessed for their GAG and collagen content both biochemically and histologically.ResultsHuman chondrocytes expanded twice as fast on devitalized synoviocyte matrix vs. tissue culture plastic, and cells retained their re-differentiation capacity for twice the number of population doublings. There was no significant difference in growth rate between low and atmospheric oxygen tension. There was significantly less collagen type I, collagen type II, aggrecan and more MMP13 expression in cells expanded on synoviocyte matrix vs. tissue culture plastic. There were also significant effects due to oxygen tension on gene expression, wherein there was greater collagen type I, collagen type II, SOX9 and less MMP13 expression on tissue culture plastic compared to synoviocyte matrix. There was a significant increase in GAG, but not collagen, accumulation in chondrocyte aggregates re-differentiated at low oxygen tension over that achieved in atmospheric oxygen conditions.ConclusionsSynoviocyte-derived matrix supports enhanced expansion of human chondrocytes such that the chondrocytes are maintained in a state from which they can re-differentiate into a cartilage phenotype after significantly more population doublings. Also, low oxygen tension supports GAG, but not collagen, accumulation. These findings are a step towards the production of a more functional, tissue engineered cartilage.     

Correlated oscillations in glucose consumption, oxygen consumption, and intracellular free Ca(2+) in single islets of Langerhans

Micron-sized sensors were used to monitor glucose and oxygen levels in the extracellular space of single islets of Langerhans in real-time. At 10 mM glucose, oscillations in intraislet glucose concentration were readily detected. Changes in glucose level correspond to changes in glucose consumption by glycolysis balanced by mass transport into the islet. Oscillations had a period of 3.1 +/- 0.2 min and amplitude of 0.8 +/- 0.1 mM glucose (n = 21). Superimposed on these oscillations were faster fluctuations in glucose level during the periods of low glucose consumption. Oxygen level oscillations that were out of phase with the glucose oscillations were also detected. Oscillations in both oxygen and glucose consumption were strongly dependent upon extracellular Ca(2+) and sensitive to nifedipine. Simultaneous measurements of glucose with intracellular Ca(2+) ([Ca(2+)](i)) revealed that decreases in [Ca(2+)](i) preceded increases in glucose consumption by 7.4 +/- 2.1 s during an oscillation (n = 9). Conversely, increases in [Ca(2+)](i) preceded increases in oxygen consumption by 1.5 +/- 0.2 s (n = 4). These results suggest that during oscillations, bursts of glycolysis begin after Ca(2+) has stopped entering the cell. Glycolysis stimulates further Ca(2+) entry, which in turn stimulates increases in respiration. The data during oscillation are in contrast to the time course of events during initial exposure to glucose. Under these conditions, a burst of oxygen consumption precedes the initial rise in [Ca(2+)](i). A model to explain these results is described.     

Role of glucose as a modulator of anabolic and catabolic gene expression in normal and osteoarthritic human chondrocytes

Cartilage matrix homeostasis involves a dynamic balance between numerous signals that modulate chondrocyte functions. This study aimed at elucidating the role of the extracellular glucose concentration in modulating anabolic and catabolic gene expression in normal and osteoarthritic (OA) human chondrocytes and its ability to modify the gene expression responses induced by pro-anabolic stimuli, namely Transforming Growth Factor-β (TGF). For this, we analyzed by real time RT-PCR the expression of articular cartilage matrix-specific and non-specific genes, namely collagen types II and I, respectively. The expression of the matrix metalloproteinases (MMPs)-1 and -13, which plays a major role in cartilage degradation in arthritic conditions, and of their tissue inhibitors (TIMP) was also measured. The results showed that exposure to high glucose (30 mM) increased the mRNA levels of both MMPs in OA chondrocytes, whereas in normal ones only MMP-1 increased. Collagen II mRNA was similarly increased in normal and OA chondrocytes, but the increase lasted longer in the later. Exposure to high glucose for 24 h prevented TGF-induced downregulation of MMP-13 gene expression in normal and OA chondrocytes, while the inhibitory effect of TGF on MMP-1 expression was only partially reduced. Other responses were not significantly modified. In conclusion, exposure of human chondrocytes to high glucose, as occurs in vivo in diabetes mellitus patients and in vitro for the production of engineered cartilage, favors the chondrocyte catabolic program. This may promote articular cartilage degradation, facilitating OA development and/or progression, as well as compromise the quality and consequent in vivo efficacy of tissue engineered cartilage.     

A bioluminescent whole-cell reporter for detection of 2, 4-dichlorophenoxyacetic acid and 2,4-dichlorophenol in soil

A bioreporter was made containing a tfdRP(DII)-luxCDABE fusion in a modified mini-Tn5 construct. When it was introduced into the chromosome of Ralstonia eutropha JMP134, the resulting strain, JMP134-32, produced a sensitive bioluminescent response to 2, 4-dichlorophenoxyacetic acid (2,4-D) at concentrations of 2.0 microM to 5.0 mM. This response was linear (R(2) = 0.9825) in the range of 2.0 microM to 1.1 x 10(2) microM. Saturation occurred at higher concentrations, with maximal bioluminescence occurring in the presence of approximately 1.2 mM 2,4-D. A sensitive response was also recorded in the presence of 2,4-dichlorophenol at concentrations below 1.1 x 10(2) microM; however, only a limited bioluminescent response was recorded in the presence of 3-chlorobenzoic acid at concentrations below 1.0 mM. A significant bioluminescent response was also recorded when strain JMP134-32 was incubated with soils containing aged 2,4-D residues.     

Vascular oxidative metabolism under different metabolic conditions.

Control of respiration in vascular smooth muscle was examined while the metabolic state of the tissue was manipulated. During KCl-induced contractures in the presence of 5 mM glucose, oxygen consumption increased by 10 nmol/per min g without any decrease in phosphocreatine (PCr) or ATP as determined by 31P-NMR indicating a control of respiration which does not involve changes in high-energy phosphates (e.g., ADP, phosphorylation potential). However, when aortae with resting tone in the absence of substrate were then provided with 5 mM 2-deoxyglucose as the sole substrate, oxygen consumption increased 7.4 nmol/min per g while PCr decreased by more than 50% (resulting in a 2-fold increase in the calculated free ADP) with no change in tension from resting tone. During a subsequent KCl induced contracture in the presence of 2-deoxyglucose, oxygen consumption increased an additional 7.2 nmol/min per g while PCr continued to decline. Therefore, at least two mechanisms of respiratory control may exist in sheep aorta, one dependent and the other independent of changes in high-energy phosphates.     

Cartilage tissue engineering: controversy in the effect of oxygen

Articular cartilage lacks the ability to repair itself and consequently defects in this tissue do not heal. Tissue engineering approaches, employing a scaffold material and cartilage producing cells (chondrocytes), hold promise for the treatment of such defects. In these strategies the limitation of nutrients, such as oxygen, during in vitro culture are of major concern and will have implications for proper bioreactor design. We recently demonstrated that oxygen gradients are indeed present within tissue engineered cartilaginous constructs. Interestingly, oxygen, besides being an essential nutrient, is also a controlling agent of developmental processes including cartilage formation. However, the specific role of oxygen in these processes is still obscure despite the recent advances in the field. In particular, the outcome of published investigations is inconsistent regarding the effect of oxygen tension on chondrocytes. Therefore, this article describes the possible roles of oxygen gradients during embryonic cartilage development and reviews the data reported on the effect of oxygen tension on in vitro chondrocyte proliferation and differentiation from a tissue engineering perspective. Furthermore, possible causes for the variance in the data are discussed. Finally, recommendations are included that may reduce the variation, resulting in more reliable and comparable data.     

A miniature Clark-type oxygen electrode using a polyelectrolyte and its application as a glucose sensor

A miniature Clark-type oxygen electrode was fabricated by anisotropically etching silicon. A two-gold-electrode configuration was used and a double-layered gas-permeable membrane was formed directly on the electrolyte, poly(vinyl-4-ethylpyridinium bromide) in the sensitive area. These materials improved the electrode's stability in long-term storage and sterilization tolerance to a practical level. The 90% response time averaged 80 s and residual current 10%, with a good linear calibration curve. The oxygen electrode was also used to make an integrated sensor for the simultaneous determination of glucose and oxygen. The glucose sensor's response time was 50-110 s, with good linearity in glucose concentrations between 56 microM and 1.1 mM at 37 degrees C, pH 7.0.     

Transmural gradients of oxygen tension on cerebral microvessels in rats

Using modified oxygen needle microelectrodes, vital microscopy with video-recording facilities, measurements of tissue oxygen tension (PO2) profiles near the cortical arterioles and transmural PO2 gradients on pial arterioles of the rat were performed. At control transmural PO2 gradient averaged 1.17 +/- 0.06 mm Hg/microm (mean +/- SEM, n = 40). Local dilatation of the arteriolar wall (microapplication of sodium nitroprusside approximately 2 x 10(-7) M) resulted in marked drop of the transmural PO2 gradient to 0.68 +/- 0.04 mm Hg/microm (p < 0.001, n = 38). The important finding of the study is the dependence of the transmural PO2 gradient on the vascular tone of pial arterioles. The data presented allow to conclude that O2 consumption of the arteriolar wall lies within the range for surrounding tissue and O2 consumption of the endothelial layer and, apparently, has no substantial impact on transmural PO2 gradient.     

Effects of oxygen on engineered cardiac muscle

Concentration gradients associated with the in vitro cultivation of engineered tissues that are vascularized in vivo result in the formation of only a thin peripheral tissue-like region (e.g., approximately 100 microm for engineered cardiac muscle) around a relatively cell-free interior. We previously demonstrated that diffusional gradients within engineered cardiac constructs can be minimized by direct perfusion of culture medium through the construct. In the present study, we measured the effects of medium perfusion rate and local oxygen concentration (p(O2)) on the in vitro reconstruction of engineered cardiac muscle. Neonatal rat cardiomyocytes were seeded onto biodegradable polymer scaffolds (fibrous discs, 1.1 cm diameter x 2 mm thick, made of polyglycolic acid, 24 x 10(6) cells per scaffold). The resulting cell-polymer constructs were cultured for a total of 12 days in serially connected cartridges (n = 1-8), each containing one construct directly perfused with culture medium at a flow rate of 0.2-3.0 mL/min. In all groups, oxygen concentration decreased due to cell respiration, and depended on construct position in the series and medium flow rate. Higher perfusion rates and higher p(O2) correlated with more aerobic cell metabolism, and higher DNA and protein contents. Constructs cultured at p(O2) of 160 mm Hg had 50% higher DNA and protein contents, markedly higher expression of sarcomeric alpha-actin, better organized sarcomeres and cell junctions, and 4.5-fold higher rate of cell respiration as compared to constructs cultured at p(O2) of 60 mm Hg. Contraction rates of the corresponding cardiac cell monolayers were 40% higher at p(O2) of 160 than 60 mm Hg. The control of oxygen concentration in cell microenvironment can thus improve the structure and function of engineered cardiac muscle. Experiments of this kind can form a basis for controlled studies of the effects of oxygen on the in vitro development of engineered tissues.     

Detection of multiple patterns of oscillatory oxygen consumption in single mouse islets of Langerhans.

A novel oxygen microsensor was used to measure oxygen levels in single mouse islets as a function of glucose concentration. Oxygen consumption of individual islets was 5.99 +/- 1.17, 9.21 +/- 2.15, and 12.22 +/- 2.16 pmol/min at 3, 10, and 20 mM glucose, respectively (mean +/- SEM, n = 10). Consumption of oxygen was islet-size dependent as larger islets consumed more oxygen than smaller islets but smaller islets consumed more oxygen per unit volume than larger islets. Elevating glucose levels from 3 to 10 mM induced pronounced fast oscillations in oxygen level (period of 12.1 +/- 1.7 s, n = 6) superimposed on top of large slow oscillations (period of 3.3 +/- 0.6 min, n = 6). The fast oscillations could be completely abolished by treatment with the L-type Ca2+-channel blocker nifedipine (40 microM) with a lesser effect on slow oscillations. Slow oscillations were almost completely dependent upon extracellular Ca2+. The oxygen patterns closely mimic those that have previously been reported for intracellular Ca2+ levels and are suggestive of an important role for Ca2+ in amplifying metabolic oscillations.