Bioengineering Cell Therapy for Treatment of Peripheral Artery Disease.

Peripheral artery disease is an atherosclerotic disease associated with limb ischemia that necessitates limb amputation in severe cases. Cell therapies comprised of adult mononuclear or stromal cells have been clinically tested and show moderate benefits. Bioengineering strategies can be applied to modify cell behavior and function in a controllable fashion. Using mechanically tunable or spatially controllable biomaterials, we highlight examples in which biomaterials can increase the survival and function of the transplanted cells to improve their revascularization efficacy in preclinical models. Biomaterials can be used in conjunction with soluble factors or genetic approaches to further modulate the behavior of transplanted cells and the locally implanted tissue environment in vivo. We critically assess the advances in bioengineering strategies such as 3-dimensional bioprinting and immunomodulatory biomaterials that can be applied to the treatment of peripheral artery disease and then discuss the current challenges and future directions in the implementation of bioengineering strategies.

Treatment of Rat Lymphedema by Propeller Lymphatic Tissue Flap Combined with Nanofibrillar Collagen Scaffolds.

BACKGROUND: The aim of our study was to evaluate a new propeller vascularized lymphatic tissue flap (pVLNT) combined with aligned nanofibrillar collagen scaffolds (CS) (BioBridge) in reducing lymphedema in the rat lymphedema model. METHODS: Unilateral left hindlimb lymphedema was created in 15 female Sprague-Dawley rats following inguinal and popliteal lymph nodes (LN) resection and radiation. An inguinal pVLNT was elevated from the contralateral groin and transferred through a skin tunnel to the affected groin. Four collagen threads were attached to the flap and inserted in the hindlimb at the subcutaneous level in a fan shape. The three study groups consisted of group A (control), group B (pVLNT), and group C (pVLNT + CS). Volumetric analysis of both hindlimbs was performed using micro-computed tomography imaging before the surgery (at initial time point) and then at 1 and 4 months, postoperatively, and the relative volume difference (excess volume) was measured for each animal. Lymphatic drainage was assessed by indocyanine green (ICG) fluoroscopy for number and morphology of new collectors and the time required for ICG to move from injection point to the midline. RESULTS: Four months after the induction of lymphedema, an increased relative volume difference remained in group A (5.32 ± 4.74%), while there was a significant relative volume reduction in group B (-13.39 ± 8.55%) and an even greater reduction in group C (-14.56 ± 5.04%). ICG fluoroscopy proved the functional restoration of lymphatic vessels and viability of pVLNT in both B and C groups. Notably, only group C demonstrated statistically significant improvements in lymphatic pattern/morphology and in the number of lymphatic collectors as compared with the control group A. CONCLUSION: The pedicle lymphatic tissue flap combined with SC is an effective procedure for the treatment of lymphedema in rats. It can be easily translated into treatment of humans' lower and upper limb lymphedema and further clinical studies are warranted.

Lymphatic regeneration after implantation of aligned nanofibrillar collagen scaffolds: Preliminary preclinical and clinical results.

BACKGROUND: We tested our hypothesis that implantation of aligned nanofibrillar collagen scaffolds (BioBridge™) can both prevent and reduce established lymphedema in the rat lymphedema model. Our authors report clinical cases that demonstrate new lymphatic formation guided by BioBridge™ as seen by near-infrared (NIR) fluoroscopy and magnetic resonance (MR) lymphography. METHODS: A rat lymphedema model was utilized. A prevention group received implantation of BioBridge™ immediately after lymphadenectomy. A lymphedema group received implantation of BioBridge™ with autologous adipose-derived stem cells (ADSC; treatment group) or remained untreated (control group). All subjects were observed for 4 months after lymphadenectomy. The hindlimb change was evaluated using computed tomography-based volumetric analysis. Lymphagiogenesis was assessed by indocyanine green (ICG) lymphography. RESULTS: Animals in the treatment group showed a reduction in affected limb volume. Animals in the prevention group showed no increase in the affected limb volume. ICG fluoroscopy demonstrated lymph flow and formation of lymphatics toward healthy lymphatics. CONCLUSIONS: In the rat lymphedema model, implantation of BioBridge™ at the time of lymph node removal prevents the development of lymphedema. Treatment of established lymphedema with the BioBridge™ and ADSC reduces lymphedema. New lymphatic vessels are demonstrated by NIR fluoroscopy and MR lymphography. These findings have implications for the treatment of lymphedema in human subjects.

Global changes to the ubiquitin system in Huntington's disease.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by expansion of CAG triplet repeats in the huntingtin (HTT) gene (also called HD) and characterized by accumulation of aggregated fragments of polyglutamine-expanded HTT protein in affected neurons. Abnormal enrichment of HD inclusion bodies with ubiquitin, a diagnostic characteristic of HD and many other neurodegenerative disorders including Alzheimer's and Parkinson's diseases, has suggested that dysfunction in ubiquitin metabolism may contribute to the pathogenesis of these diseases. Because modification of proteins with polyubiquitin chains regulates many essential cellular processes including protein degradation, cell cycle, transcription, DNA repair and membrane trafficking, disrupted ubiquitin signalling is likely to have broad consequences for neuronal function and survival. Although ubiquitin-dependent protein degradation is impaired in cell-culture models of HD and of other neurodegenerative diseases, it has not been possible to evaluate the function of the ubiquitin-proteasome system (UPS) in HD patients or in animal models of the disease, and a functional role for UPS impairment in neurodegenerative disease pathogenesis remains controversial. Here we exploit a mass-spectrometry-based method to quantify polyubiquitin chains and demonstrate that the abundance of these chains is a faithful endogenous biomarker of UPS function. Lys 48-linked polyubiquitin chains accumulate early in pathogenesis in brains from the R6/2 transgenic mouse model of HD, from a knock-in model of HD and from human HD patients, establishing that UPS dysfunction is a consistent feature of HD pathology. Lys 63- and Lys 11-linked polyubiquitin chains, which are not typically associated with proteasomal targeting, also accumulate in the R6/2 mouse brain. Thus, HD is linked to global changes in the ubiquitin system to a much greater extent than previously recognized.

Biomedical Applications of Collagen.

Extracellular matrix proteins (ECMs) provide structural support and dynamic signaling cues that regulate cell behavior and tissue morphogenesis [...].

Development of a rat model of lymphedema and the implantation of a collagen-based medical device for therapeutic intervention.

Secondary lymphedema is a common condition among cancer survivors, and treatment strategies to prevent or treat lymphedema are in high demand. The development of novel strategies to diagnose or treat lymphedema would benefit from a robust experimental animal model of secondary lymphedema. The purpose of this methods paper is to describe and summarize our experience in developing and characterizing a rat hindlimb model of lymphedema. Here we describe a protocol to induce secondary lymphedema that takes advantage of micro computed tomography imaging for limb volume measurements and visualization of lymph drainage with near infrared imaging. To demonstrate the utility of this preclinical model for studying the therapeutic benefit of novel devices, we apply this animal model to test the efficacy of a biomaterials-based implantable medical device.

A Biomimetic High Throughput Model of Cancer Cell Spheroid Dissemination onto Aligned Fibrillar Collagen.

Cell dissemination during tumor development is a characteristic of cancer metastasis. Dissemination from three-dimensional spheroid models on extracellular matrices designed to mimic tissue-specific physiological microenvironments may allow us to better elucidate the mechanism behind cancer metastasis and the response to therapeutic agents. The orientation of fibrillar collagen plays a key role in cellular processes and mediates metastasis through contact-guidance. Understanding how cells migrate on aligned collagen fibrils requires in vitro assays with reproducible and standardized orientation of collagen fibrils on the macro-to-nanoscale. Herein, we implement a spheroid-based migration assay, integrated with a fibrillar type I collagen matrix, in a manner compatible with high throughput image acquisition and quantitative analysis. The migration of highly proliferating U2OS osteosarcoma cell spheroids onto an aligned fibrillar type I collagen matrix was quantified. Cell dissemination from the spheroid was polarized with increased invasion in the direction of fibril alignment. The resulting area of cell dissemination had an aspect ratio of 1.2 ± 0.1 and an angle of maximum invasion distance of 5° ± 44° relative to the direction of collagen fibril alignment. The assay described here can be applied to a fully automated imaging and analysis pipeline for the assessment of tumor cell migration with high throughput screening.

Delivery of Human Stromal Vascular Fraction Cells on Nanofibrillar Scaffolds for Treatment of Peripheral Arterial Disease.

Cell therapy for treatment of peripheral arterial disease (PAD) is a promising approach but is limited by poor cell survival when cells are delivered using saline. The objective of this study was to examine the feasibility of aligned nanofibrillar scaffolds as a vehicle for the delivery of human stromal vascular fraction (SVF), and then to assess the efficacy of the cell-seeded scaffolds in a murine model of PAD. Flow cytometric analysis was performed to characterize the phenotype of SVF cells from freshly isolated lipoaspirate, as well as after attachment onto aligned nanofibrillar scaffolds. Flow cytometry results demonstrated that the SVF consisted of 33.1 ± 9.6% CD45+ cells, a small fraction of CD45-/CD31+ (4.5 ± 3.1%) and 45.4 ± 20.0% of CD45-/CD31-/CD34+ cells. Although the subpopulations of SVF did not change significantly after attachment to the aligned nanofibrillar scaffolds, protein secretion of vascular endothelial growth factor (VEGF) significantly increased by six-fold, compared to SVF cultured in suspension. Importantly, when SVF-seeded scaffolds were transplanted into immunodeficient mice with induced hindlimb ischemia, the cell-seeded scaffolds induced a significant higher mean perfusion ratio after 14 days, compared to cells delivered using saline. Together, these results show that aligned nanofibrillar scaffolds promoted cellular attachment, enhanced the secretion of VEGF from attached SVF cells, and their implantation with attached SVF cells stimulated blood perfusion recovery. These findings have important therapeutic implications for the treatment of PAD using SVF.

Delivery of hepatocyte growth factor mRNA from nanofibrillar scaffolds in a pig model of peripheral arterial disease.

Background: Chemical modification of mRNA (mmRNA) substantially improves their stability and translational efficiency within cells. Nanofibrillar collagen scaffolds were previously shown to enable the spatially localized delivery and temporally controlled release of mmRNA encoding HGF both in vitro and in vivo. Materials & methods: Herein we developed an improved slow-releasing HGF mmRNA scaffold and tested its therapeutic efficacy in a porcine model of peripheral arterial disease. Results & conclusion: The HGF mmRNA was released from scaffolds in a temporally controlled fashion in vitro with preserved transfection activity. The mmRNA scaffolds improved vascular regeneration when sutured to the ligated porcine femoral artery. These studies validate the therapeutic potential of HGF mmRNA delivery from nanofibrillar scaffolds for treatment of peripheral arterial disease.

Aligned Nanofibrillar Scaffolds for Controlled Delivery of Modified mRNA.

RNA-based vector delivery is a promising gene therapy approach. Recent advances in chemical modification of mRNA structure to form modified mRNA (mmRNA or cmRNA or modRNA) have substantially improved their stability and translational efficiency within cells. However, mmRNA conventionally delivered in solution can be taken up nonspecifically or become cleared away prematurely, which markedly limits the potential benefit of mmRNA therapy. To address this limitation, we developed mmRNA-incorporated nanofibrillar scaffolds that could target spatially localized delivery and temporally controlled release of the mmRNA both in vitro and in vivo. To establish the efficacy of mmRNA therapy, mmRNA encoding reporter proteins such as green fluorescence protein or firefly luciferase (Fluc) was loaded into aligned nanofibrillar collagen scaffolds. The mmRNA was released from mmRNA-loaded scaffolds in a transient and temporally controlled manner and induced transfection of human fibroblasts in a dose-dependent manner. In vitro transfection was further verified using mmRNA encoding the angiogenic growth factor, hepatocyte growth factor (HGF). Finally, scaffold-based delivery of HGF mmRNA to the site of surgically induced muscle injury in mice resulted in significantly higher vascular regeneration after 14 days, compared to implantation of Fluc mmRNA-releasing scaffolds. After transfection with Fluc mmRNA-releasing scaffold in vivo, Fluc activity was detectable and localized to the muscle region, based on noninvasive bioluminescence imaging. Scaffold-based local mmRNA delivery as an off-the-shelf form of gene therapy has broad translatability for treating a wide range of diseases or injuries.

Vaccine Prophylaxis of Pneumococcal Infections in Children under Conditions of Severe Flood in the Amur River Basin.

BACKGROUND: Pneumococcal infection being one of the dominant causes of acute respiratory diseases and exacerbation of chronic ones is a serious problem for human health and society. The flood in the Amur river basin in the summer of 2013 created a special zone and risk conditions for the formation of respiratory pathology in the Far-Eastern region of Russia. We aimed to give clinical and epidemiological assessment of the effectiveness of vaccination programs of respiratory viral and pneumococcal infections and generalization of regional experience in the organization of a set of measures aimed at their prevention in the postflood period in the Far-Eastern region. METHODS: The monitoring program includes children aged 2 to 5 years in the amount of 4988 with risk factors for pneumococcal infection. The pneumococcal conjugate vaccine Prevenar-13 was used for immunization. Data on the incidence of ARVI and pneumonia in children in pre- and postvaccination periods were to be recorded. The indicators and special criteria were used to assess the effectiveness of vaccination. To study the circulation of serovariants of pneumococcus in inflammatory diseases of the respiratory tract and nasopharyngeal carrier, bacteriological and molecular genetic methods (RT-PCR in the mode of multiprime detection) were used. RESULTS: Differences in the frequency and range of serovariants of circulating isolates of pneumococcus in the postvaccinal period and in unvaccinated children, elimination of a number of serotypes, and appearance of circulation of nonvaccinated strains were revealed. The incidence of acute respiratory diseases and pneumonia among the vaccinated population for 2 years in the region decreased by 2.5 times. The coefficient of effectiveness of vaccination according to the indicator of morbidity of children with pneumonia reaches 75-100% with direct dependence on the age of children (r=0.98). CONCLUSION: Comparative statistical analysis revealed a high degree of effectiveness of regional programs with the methods of immunoprophylaxis of pneumococcal infections.

Brain oxygenation and metabolism during selective cerebral perfusion in neonates.

OBJECTIVE: To investigate the possible neuroprotective effects of selective cerebral perfusion (SCP) during deep hypothermic circulatory arrest on brain oxygenation and metabolism in newborn piglets. METHODS: Newborn piglets 2-4 days of age, anesthetized and mechanically ventilated, were used for the study. The animals were placed on cardiopulmonary bypass, cooled to 18 degrees C and put on SCP (20 ml/(kg min)) for 90 min. After rewarming, the animals were monitored through 2h of recovery. Oxygen pressure in the microvasculature of the cortex was measured by oxygen-dependent quenching of phosphorescence. The extracellular level of dopamine in striatum was measured by microdialysis and hydroxyl radicals by ortho-tyrosine levels. Levels of phosphorylated cAMP response element binding protein (pCREB) in striatal tissue were measured by Western blots using antibodies specific for phosphorylated CREB. The results are presented as mean+/-SD (p<0.05 was significant). RESULTS: Pre-bypass cortical oxygen pressure was 48.9+/-11.3 mmHg and during the first 5 min of SCP, the peak of the histogram, corrected to 18 degrees C, decreased to 11.2+/-3.8 mmHg (p<0.001) and stayed near that value to the end of bypass. The mean value for the peak of the histograms measured at the end of SCP was 8+/-3 mmHg (p<0.001). SCP completely prevented the deep hypothermic circulatory arrest-dependent increase in extracellular dopamine and hydroxyl radicals. After SCP, there was a statistically significant increase in pCREB immunoreactivity (534+/-60%) compared to the sham-operated group (100+/-63%, p<0.005). Measurements of total CREB showed that SCP did induce a statistically significant increase in CREB as compared to sham-operated animals (168+/-31%, p<0.05). CONCLUSION: SCP, as compared to DHCA, improved cortical oxygenation and prevented increases in the extracellular dopamine and hydroxyl radicals. The increase in pCREB in the striatum following SCP may contribute to improved cellular recovery after this procedure.

Aligned nanofibrillar collagen scaffolds - Guiding lymphangiogenesis for treatment of acquired lymphedema.

Secondary lymphedema is a common disorder associated with acquired functional impairment of the lymphatic system. The goal of this study was to evaluate the therapeutic efficacy of aligned nanofibrillar collagen scaffolds (BioBridge) positioned across the area of lymphatic obstruction in guiding lymphatic regeneration. In a porcine model of acquired lymphedema, animals were treated with BioBridge scaffolds, alone or in conjunction with autologous lymph node transfer as a source of endogenous lymphatic growth factor. They were compared with a surgical control group and a second control group in which the implanted BioBridge was supplemented with exogenous vascular endothelial growth factor-C (VEGF-C). Three months after implantation, immunofluorescence staining of lymphatic vessels demonstrated a significant increase in lymphatic collectors within close proximity to the scaffolds. To quantify the functional impact of scaffold implantation, bioimpedance was used as an early indicator of extracellular fluid accumulation. In comparison to the levels prior to implantation, the bioimpedance ratio was significantly improved only in the experimental BioBridge recipients with or without lymph node transfer, suggesting restoration of functional lymphatic drainage. These results further correlated with quantifiable lymphatic collectors, as visualized by contrast-enhanced computed tomography. They demonstrate the therapeutic potential of BioBridge scaffolds in secondary lymphedema.

CREB phosphorylation following hypoxia and ischemia in striatum of newborn piglets: possible role of dopamine.

The goal of the present study was to determine the effects of hypoxia and ischemia and the role of dopamine on phosphorylation of cAMP response element binding protein (CREB) in striatum of newborn piglets. Piglets, with and without prior injection of alpha-methyl-p-tyrosine (AMT), an inhibitor of dopamine (DA) synthesis, were subjected to 1 h of hypoxia (decreased inspired oxygen pressure, FiO2, from 21 to 6%) or 1 h of ischemia (ligation of both carotid arteries and hemorrhage to reduce the systemic arterial pressure to about 40 mmHg), followed by 2 h recovery. Microvascular oxygen pressure in the cortex (pCO2) was measured by quenching of phosphorescence. Extracellular DA was determined by in vivo microdialysis. Striatal levels of phosphorylated CREB (pCREB) and total CREB were determined by Western blots. In sham-operated animals, pCO2 was 49.7 +/- 8.2 mmHg. During hypoxia and ischemia, pCO2 decreased to 6.3 +/- 1.8 mmHg and 10.2 +/- 2.7 mmHg, respectively. There was statistical difference in the level of extracellular DA during hypoxia versus ischemia. At the end of ischemia and hypoxia, the levels of DA were 96 x 10(3) +/- 24 x 10(3)% and 26 x 10(3) +/- 12 x 10(3)% of control, respectively. The pCREB measured after 2 h recovery was not changed after hypoxia but was decreased to 47.8 +/- 24% of control after ischemia. Depletion of endogenous DA abolished the ischemia-induced decrease in pCREB level. Total CREB did not change after either condition. It can be concluded that observed decreases of CREB phosphorylation following ischemia can be at least partially due to the high extracellular DA level.

Cerebral oxygenation during repetitive apnea in newborn piglets.

This study examined the effect of repetitive apnea on brain oxygen pressure in newborn piglets. Each animal was given 10 episodes of apnea, initiated by disconnecting them from the ventilator and completed by reconnecting them to the ventilation circuit. The apneic episodes were ended 30 sec after the heart rate reached the bradycardic threshold of 60 beats per min. The oxygen pressure in the microvasculature of the cortex was measured by oxygen-dependent quenching of the phosphorescence. In all experiments, the blood pressure, body temperature, and heart rate were continuously monitored. Arterial blood samples were taken throughout the experiment and the blood pH, PaO2 and PaCO2 were measured. During pre-apnea, cortical oxygen was 55.1 +/- 6.4 (SEM, n = 7) mm Hg and decreased during each apnea to 8.1 +/- 2.8 mm Hg. However, the values of cortical oxygen varied during recovery periods. Maximal oxygen levels during recovery from the first two apneic episodes were 76.8 +/- 12 mm Hg and 69.6 +/- 9 mm Hg, respectively, values higher than pre-apnea. Cortical oxygen pressure then progressively decreased following consequent apnea. In conclusion, the data show that repetitive apnea caused a progressive decrease in cortical oxygen levels in the brain of newborn piglets. This deficit in brain oxygenation can be at least partly responsible for the neurological side effects of repetitive apnea.

Aligned-Braided Nanofibrillar Scaffold with Endothelial Cells Enhances Arteriogenesis.

The objective of this study was to enhance the angiogenic capacity of endothelial cells (ECs) using nanoscale signaling cues from aligned nanofibrillar scaffolds in the setting of tissue ischemia. Thread-like nanofibrillar scaffolds with porous structure were fabricated from aligned-braided membranes generated under shear from liquid crystal collagen solution. Human ECs showed greater outgrowth from aligned scaffolds than from nonpatterned scaffolds. Integrin α1 was in part responsible for the enhanced cellular outgrowth on aligned nanofibrillar scaffolds, as the effect was abrogated by integrin α1 inhibition. To test the efficacy of EC-seeded aligned nanofibrillar scaffolds in improving neovascularization in vivo, the ischemic limbs of mice were treated with EC-seeded aligned nanofibrillar scaffold; EC-seeded nonpatterned scaffold; ECs in saline; aligned nanofibrillar scaffold alone; or no treatment. After 14 days, laser Doppler blood spectroscopy demonstrated significant improvement in blood perfusion recovery when treated with EC-seeded aligned nanofibrillar scaffolds, in comparison to ECs in saline or no treatment. In ischemic hindlimbs treated with scaffolds seeded with human ECs derived from induced pluripotent stem cells (iPSC-ECs), single-walled carbon nanotube (SWNT) fluorophores were systemically delivered to quantify microvascular density after 28 days. Near infrared-II (NIR-II, 1000-1700 nm) imaging of SWNT fluorophores demonstrated that iPSC-EC-seeded aligned scaffolds group showed significantly higher microvascular density than the saline or cells groups. These data suggest that treatment with EC-seeded aligned nanofibrillar scaffolds improved blood perfusion and arteriogenesis, when compared to treatment with cells alone or scaffold alone, and have important implications in the design of therapeutic cell delivery strategies.

Comparison of low-flow cardiopulmonary bypass and circulatory arrest on brain oxygen and metabolism.

BACKGROUND: In the neonatal brain we measured oxygen (Bo(2)), extracellular striatal dopamine (DA), and striatal tissue levels of ortho-tyrosine (o-tyr) during low-flow cardiopulmonary bypass (LFCPB) or deep hypothermic circulatory arrest (DHCA) and the post-bypass recovery period. METHODS: Newborn piglets were assigned to sham (n = 6), LFCPB (n = 8), or DHCA (n = 6) groups. Animals were cooled to 18 degrees C and underwent DHCA or LFCPB (20 mL x kg(-1) x min(-1)) for 90 minutes. The Bo(2) was measured by quenching the phosphorescence, DA by microdialysis, and hydroxyl radicals by o-tyr levels. The results are presented as the mean +/- SD (p < 0.05 was significant). RESULTS: Baseline Bo(2) was between 45 to 60 mm Hg. At the end of LFCPB, Bo(2) was 10.5 +/- 1.2 mm Hg. By 5 and 30 minutes of arrest during DHCA, Bo(2) fell to 4.2 +/- 2.5 mm Hg and 1.4 +/- 0.7 mm Hg, respectively. Compared with control, extracellular DA did not change during LFCPB. During DHCA extracellular levels of DA increased, by 750-fold from baseline at 45 minutes and to a maximum of 53000-fold at 75 minutes. After 2 hours of recovery from DHCA, the o-tyr within the striatum increased about sixfold as compared with control. There was no change in o-tyr measured after LFCPB. CONCLUSIONS: In DHCA, but not LFCPB, levels of DA and o-tyr increased considerably in the striatum of piglets, a finding that may indicate the exhaustion of cellular energy levels and contribute substantially to cellular injury.

Brain oxygenation during cardiopulmonary bypass and circulatory arrest.

Quantitative measurements of oxygen distribution in the microcirculation of the brain cortex of newborn piglets were made during different modes of cardiopulmonary bypass. Three groups of animals, anesthetized and mechanically ventilated, were studied. The first group of animals were maintained on normothermic cardiopulmonary bypass (CPB) at a flow of 100 ml/kg/min, while the second and third groups underwent low flow hypothermic cardiopulmonary bypass (40 ml/kg/min at 18 degrees C) (LFCPB) and deep hypothermic (18 degrees C) circulatory arrest (DHCA), respectively. After bypass, the piglets were monitored for a two hours post-bypass recovery period. CPB caused a decrease in the cortical oxygen from 62 +/- 3 mm Hg to 32 +/- 7 mm Hg at the beginning of bypass and to 36 +/- 5 mm Hg at the end of bypass. During the recovery period, cortical oxygenation steadily decreased, reaching 29 +/- 8 mm Hg at the end of the experiment. With initiation of LFCPB, cortical oxygen decreased to 22 +/- 7 mm Hg. Upon rewarming cortical oxygen increased to 37 +/- 5 mm Hg and then decreased again to about 30 mm Hg at the end of two hours of post-bypass recovery. Similar changes in cortical oxygenation were observed during DHCA. In DHCA cortical oxygen decreased to 19 +/- 4 mm Hg and during rewarming and recovery increased to 35 +/- 6 mm Hg. In conclusion, it has been shown that in newborn piglets recovering from CPB, LFCPB and DHCA, when the blood pressure remained above 55 mm Hg and therefore total blood flow should be well maintained, oxygen pressure in the microvasculature is significantly lower than for pre-bypass. It is suggested that the decreased oxygenation is due to increased heterogeneity in resistance in the microcirculatory units, resulting in broadened distribution of flow rates and oxygen levels.

Altered gene expression following cardiopulmonary bypass and circulatory arrest.

This study investigated the effects of normothermic cardiopulmonary bypass (CPB) and circulatory arrest (DHCA) on expression of specific genes in neonatal piglet brain. CPB was performed through the chest at 100 ml/kg/min for 2 hrs at 37 degrees C. In the second group of animals, CPB was begun as described above and then animals were cooled to a nasopharyngeal/brain temperature of 18 degrees C. When the brain temperature reached 18 degrees C, the CPB circuit was turned off. After 60 min of circulatory arrest (DHCA), CPB was resumed at 100 ml/kg/min, and the piglets were rewarmed to a temperature of 36 degrees C. In both groups, the animals remain sedated, paralyzed, mechanically ventilated, and continuously monitored throughout a four hour study period after CPB. Oxygen pressure in the microvasculature of the cortex was measured by oxygen dependent quenching of phosphorescence. The aRNA technique was used to assess mRNA steady-state levels in the brain tissue. Control oxygen pressure (pre-bypass) was 61 +/- 5 Torr and during CPB this decreased to 32 +/- 7 Torr on the beginning of bypass and to 36 +/- 5 Torr at the end of bypass. During the recovery period, cortical oxygenation steadily decreased, reaching 29 +/- 8 Torr at the end of the four hours period. Cortical oxygen decreased during DHCA to near zero and during rewarming and recovery increased to 35 +/- 6 Torr. Measurements of gene expression following CPB revealed significantly increased levels of mRNA for NMDAR1, DARPP-32, CamKII, GluR1, and D1AR. DHCA caused changes similar to those for CPB in levels of mRNA for NMDAR1, DARPP-32, CamKII and GluR1. In contrast, DHCA caused significantly increased levels of mRNA for GluR6 and GABRB1. There was no significant alteration in the level of D1AR following DHCA. The results showed that DHCA caused much larger alterations in gene expression in the critical metabolic signaling pathways tested than did CPB.

Effect of collagen nanotopography on keloid fibroblast proliferation and matrix synthesis: implications for dermal wound healing.

Keloids are locally exuberant dermal scars characterized by excessive fibroblast proliferation and matrix accumulation. Although treatment strategies include surgical removal and intralesional steroid injections, an effective regimen is yet to be established due to a high rate of recurrence. The regressing center and growing margin of the keloid have different collagen architecture and also differ in the rate of proliferation. To investigate whether proliferation is responsive to collagen topography, keloid, scar, and dermal fibroblasts were cultured on nanopatterned scaffolds varying in collagen fibril diameter and alignment-small and large diameter, aligned and random fibrils, and compared to cells grown on flat collagen-coated substrates, respectively. Cell morphology, proliferation, and expression of six genes related to proliferation (cyclin D1), phenotype (α-smooth muscle actin), and matrix synthesis (collagens I and III, and matrix metalloproteinase-1 and -2) were measured to evaluate cell response. Fibril alignment was shown to reduce proliferation and matrix synthesis in all three types of fibroblasts. Further, keloid cells were found to be most responsive to nanotopography.