Effects of cerebral perfusion pressure and increased fraction of inspired oxygen on brain tissue oxygen, lactate and glucose in patients with severe head injury

OBJECTIVE: The purpose of the study was to measure the effects of increased inspired oxygen on patients suffering severe head injury and consequent influences on the correlations between CPP and brain tissue oxygen (PtiO2) and the effects on brain microdialysate glucose and lactate. METHODS: In a prospective, observational study 20 patients suffering severe head injury (GCS< or =8) were studied between January 2000 and December 2001. Each patient received an intraparenchymal ICP device and an oxygen sensor and, in 17 patients brain microdialysis was performed at the cortical-subcortical junction. A 6 h 100% oxygen challenge (F IO2 1.0) ( Period A) was performed as early as possible in the first 24 hours after injury and compared with a similar 6 hour period following the challenge ( Period B). Statistics were performed using the linear correlation analysis, one sample t-test, as well as the Lorentzian peak correlation analysis. RESULTS: F IO2 was positively correlated with PtiO2 (p < 0.0001) over the whole study period. PtiO2 was significantly higher (p < 0.001) during Period A compared to Period B. CPP was positively correlated with PtiO2 (p < 0.001) during the whole study. PtiO2 peaked at a CPP value of 78 mmHg performing a Lorentzian peak correlation analysis of all patients over the whole study. During Period A the brain microdialysate lactate was significantly lower (p = 0.015) compared with Period B. However the brain microdialysate glucose remained unchanged. CONCLUSION: PtiO2 is significantly positively correlated with F IO2, meaning that PtiO2 can be improved by the simple manipulation of increasing F IO2 and ABGAO2. PtiO2 is positively correlated with CPP, peaking at a CPP value of 78 mmHg. Brain microdialysate lactate can be lowered by increasing PtiO2 values, as observed during the oxygen challenge, whereas microdialysate glucose is unchanged during this procedure. Extension of the oxygen challenge time and measurement of the intermediate energy metabolite pyruvate may clarify the metabolic effects of the intervention. Prospective comparative studies, including analysis of outcome on a larger multicenter basis, are necessary to assess the long term clinical benefits of this procedure.

Use of a 3D perfusion bioreactor with osteoblasts and osteoblast/endothelial cell co-cultures to improve tissue-engineered bone

The delivery of oxygen, nutrients, and the removal of waste are essential for cellular survival. Culture systems for 3D bone tissue engineering have addressed this issue by utilizing perfusion flow bioreactors that stimulate osteogenic activity through the delivery of oxygen and nutrients by low-shear fluid flow. It is also well established that bone responds to mechanical stimulation, but may desensitize under continuous loading. While perfusion flow and mechanical stimulation are used to increase cellular survival in vitro, 3D tissue-engineered constructs face additional limitations upon in vivo implantation. As it requires significant amounts of time for vascular infiltration by the host, implants are subject to an increased risk of necrosis. One solution is to introduce tissue-engineered bone that has been pre-vascularized through the co-culture of osteoblasts and endothelial cells on 3D constructs. It is unclear from previous studies: 1) how 3D bone tissue constructs will respond to partitioned mechanical stimulation, 2) how gene expression compares in 2D and in 3D, 3) how co-cultures will affect osteoblast activity, and 4) how perfusion flow will affect co-cultures of osteoblasts and endothelial cells. We have used an integrated approach to address these questions by utilizing mechanical stimulation, perfusion flow, and a co-culture technique to increase the success of 3D bone tissue engineering. We measured gene expression of several osteogenic and angiogenic genes in both 2D and 3D (static culture and mechanical stimulation), as well as in 3D cultures subjected to perfusion flow, mechanical stimulation and partitioned mechanical stimulation. Finally, we co-cultured osteoblasts and endothelial cells on 3D scaffolds and subjected them to long-term incubation in either static culture or under perfusion flow to determine changes in gene expression as well as histological measures of osteogenic and angiogenic activity. We discovered that 2D and 3D osteoblast cultures react differently to shear stress, and that partitioning mechanical stimulation does not affect gene expression in our model. Furthermore, our results suggest that perfusion flow may rescue 3D tissue-engineered constructs from hypoxic-like conditions by reducing hypoxia-specific gene expression and increasing histological indices of both osteogenic and angiogenic activity. Future research to elucidate the mechanisms behind these results may contribute to a more mature bone-like structure that integrates more quickly into host tissue, increasing the potential of bone tissue engineering.

Tolerance of human placental tissue to severe hypoxia and its relevance for dual ex vivo perfusion

In the dual ex vivo perfusion of an isolated human placental cotyledon it takes on average 20-30 min to set up stable perfusion circuits for the maternal and fetal vascular compartments. In vivo placental tissue of all species maintains a highly active metabolism and it continues to puzzle investigators how this tissue can survive 30 min of ischemia with more or less complete anoxia following expulsion of the organ from the uterus and do so without severe damage. There seem to be parallels between "depressed metabolism" seen in the fetus and the immature neonate in the peripartum period and survival strategies described in mammals with increased tolerance of severe hypoxia like hibernators in the state of torpor or deep sea diving turtles. Increased tolerance of hypoxia in both is explained by "partial metabolic arrest" in the sense of a temporary suspension of Kleiber's rule. Furthermore the fetus can react to major changes in surrounding oxygen tension by decreasing or increasing the rate of specific basal metabolism, providing protection against severe hypoxia as well as oxidative stress. There is some evidence that adaptive mechanisms allowing increased tolerance of severe hypoxia in the fetus or immature neonate can also be found in placental tissue, of which at least the villous portion is of fetal origin. A better understanding of the molecular details of reprogramming of fetal and placental tissues in late pregnancy may be of clinical relevance for an improved risk assessment of the individual fetus during the critical transition from intrauterine life to the outside and for the development of potential prophylactic measures against severe ante- or intrapartum hypoxia. Responses of the tissue to reperfusion deserve intensive study, since they may provide a rational basis for preventive measures against reperfusion injury and related oxidative stress. Modification of the handling of placental tissue during postpartum ischemia, and adaptation of the artificial reperfusion, may lead to an improvement of the ex vivo perfusion technique.

Flow modeling in a novel non-perfusion conical bioreactor

We have developed a bioreactor vessel design which has the advantages of simplicity and ease of assembly and disassembly, and with the appropriately determined flow rate, even allows for a scaffold to be suspended freely regardless of its weight. This article reports our experimental and numerical investigations to evaluate the performance of a newly developed non-perfusion conical bioreactor by visualizing the flow through scaffolds with 45° and 90° fiber lay down patterns. The experiments were conducted at the Reynolds numbers (Re) 121, 170, and 218 based on the local velocity and width of scaffolds. The flow fields were captured using short-time exposures of 60 µm particles suspended in the bioreactor and illuminated using a thin laser sheet. The effects of scaffold fiber lay down pattern and Reynolds number were obtained and correspondingly compared to results obtained from a computational fluid dynamics (CFD) software package. The objectives of this article are twofold: to investigate the hypothesis that there may be an insufficient exchange of medium within the interior of the scaffold when using our non-perfusion bioreactor, and second, to compare the flows within and around scaffolds of 45° and 90° fiber lay down patterns. Scaffold porosity was also found to influence flow patterns. It was therefore shown that fluidic transport could be achieved within scaffolds with our bioreactor design, being a non-perfusion vessel. Fluid velocities were generally same of the same or one order lower in magnitude as compared to the inlet flow velocity. Additionally, the 90° fiber lay down pattern scaffold was found to allow for slightly higher fluid velocities within, as compared to the 45° fiber lay down pattern scaffold. This was due to the architecture and pore arrangement of the 90° fiber lay down pattern scaffold, which allows for fluid to flow directly through (channel-like flow).

Perfusion model system to generate engineered grafts with controlled cellular distributions

Engineered tissue grafts, which mimic the spatial variations of cell density and extracellular matrix present in native tissues, could facilitate more efficient tissue regeneration and integration. We previously demonstrated that cells could be uniformly seeded throughout a 3D scaffold having a random pore architecture using a perfusion bioreactor2. In this work, we aimed to generate 3D constructs with defined cell distributions based on rapid prototyped scaffolds manufactured with a controlled gradient in porosity. Computational models were developed to assess the influence of fluid flow, associated with pore architecture and perfusion regime, on the resulting cell distribution.

A collagen network phase improves cell seeding of open-pore structure scaffolds under perfusion

Scaffolds with open-pore morphologies offer several advantages in cell-based tissue engineering, but their use is limited by a low cell seeding efficiency. We hypothesized that inclusion of a collagen network as filling material within the open-pore architecture of polycaprolactone-tricalcium phosphate (PCL-TCP) scaffolds increases human bone marrow stromal cells (hBMSC) seeding efficiency under perfusion and in vivo osteogenic capacity of the resulting constructs. PCL-TCP scaffolds, rapid prototyped with a honeycomb-like architecture, were filled with a collagen gel and subsequently lyophilized, with or without final crosslinking. Collagen-free scaffolds were used as controls. The seeding efficiency was assessed after overnight perfusion of expanded hBMSC directly through the scaffold pores using a bioreactor system. By seeding and culturing freshly harvested hBMSC under perfusion for 3 weeks, the osteogenic capacity of generated constructs was tested by ectopic implantation in nude mice. The presence of the collagen network, independently of the crosslinking process, significantly increased the cell seeding efficiency (2.5-fold), and reduced the loss of clonogenic cells in the supernatant. Although no implant generated frank bone tissue, possibly due to the mineral distribution within the scaffold polymer phase, the presence of a non crosslinked collagen phase led to in vivo formation of scattered structures of dense osteoids. Our findings verify that the inclusion of a collagen network within open morphology porous scaffolds improves cell retention under perfusion seeding. In the context of cell-based therapies, collagen-filled porous scaffolds are expected to yield superior cell utilization, and could be combined with perfusion-based bioreactor devices to streamline graft manufacture.

Can vasculature changes be characterised morphologically after closed soft tissue trauma?

INTRODUCTION Closed soft tissue trauma (CSTT) can be the result of a blunt impact, or a prolonged crush injury and involves damage to the skin, muscles and the neurovascular system. It causes a variety of symptoms such as haematoma and in severe cases may result in hypoxia and necrosis. There is evidence that early vasculature changes following the injury delays the tissue healing [1]. However, a precise qualitative and quantitative morphological assessment of vasculature changes after trauma and the effect of this on CSTT healing is currently missing. Research aims: Developing an experimental rat model to characterise the structural changes to the vasculature after trauma qualitatively and quantitatively using micro CT. MATERIAL AND METHODS An impact device was developed to apply a controlled reproducible CSTT to the left thigh (Biceps Femoris) of anaesthetised rats [3]. After euthanizing the animals at 6 hours after trauma, CSTT was qualitatively evaluated by macroscopic observations of the skin and muscles. For vasculature visualisation, the blood vessels of sacrificed rats were flushed with heparinised saline and then perfused with a radio-opaque contrast agent (Microfil) using an infusion pump (Figure 4). The overall changes to the vasculature as a result of impact trauma were characterised qualitatively based on the 3D reconstructed images of the vasculature (Figure 5). For a smaller region of interest, the morphological parameters such as vessel thickness (diameter), spacing, and average number per volume were quantified using the scanner’s software. RESULTS AND DISCUSSION Visual observation of CSTT has revealed a haematoma in some animals (Figure 3). Micro CT images indicate good perfusion of the vasculature with contrast agent, allowing the major vessels to be identified (Figure 5). Qualitatively and quantitatively, no differences between injured and non-injured legs were observed at 6 h after trauma. Further time points of 12h, 24h, 3 days and 14 days after trauma will be characterised for identifying temporal changes of the vasculature during healing. Histomorphometical studies are required for validation of the results derived from the micro CT imaging. CONCLUSION AND FUTURE DIRECTION Findings of this research may contribute towards the establishment of a fundamental basis for the quantitative assessment and monitoring of CSTT based on microvasculature changes after trauma, which will ultimately allow for optimising the clinical treatment and improve patient outcomes.

Effects of scaffold architecture on mechanical characteristics and osteoblast response to static and perfusion bioreactor cultures

Tissue engineering focuses on the repair and regeneration of tissues through the use of biodegradable scaffold systems that structurally support regions of injury whilst recruiting and/or stimulating cell populations to rebuild the target tissue. Within bone tissue engineering, the effects of scaffold architecture on cellular response have not been conclusively characterized in a controlled-density environment. We present a theoretical and practical assessment of the effects of polycaprolactone (PCL) scaffold architectural modifications on mechanical and flow characteristics as well as MC3T3-E1 preosteoblast cellular response in an in vitro static plate and custom-designed perfusion bioreactor model. Four scaffold architectures were contrasted, which varied in inter-layer lay-down angle and offset between layers, whilst maintaining a structural porosity of 60 ± 5%. We established that as layer angle was decreased (90° vs. 60°) and offset was introduced (0 vs. 0.5 between layers), structural stiffness, yield stress, strength, pore size and permeability decreased, whilst computational fluid dynamics-modeled wall shear stress was increased. Most significant effects were noted with layer offset. Seeding efficiencies in static culture were also dramatically increased due to offset (~45% to ~86%), with static culture exhibiting a much higher seeding efficiency than perfusion culture. Scaffold architecture had minimal effect on cell response in static culture. However, architecture influenced osteogenic differentiation in perfusion culture, likely by modifying the microfluidic environment.

Whole-Body Imaging with Single-Cell Resolution by Tissue Decolorization

The development of whole-body imaging at single-cell resolution enables system-level approaches to studying cellular circuits in organisms. Previous clearing methods focused on homogenizing mismatched refractive indices of individual tissues, enabling reductions in opacity but falling short of achieving transparency. Here, we show that an aminoalcohol decolorizes blood by efficiently eluting the heme chromophore from hemoglobin. Direct transcardial perfusion of an aminoalcohol-containing cocktail that we previously termed CUBIC coupled with a 10 day to 2 week clearing protocol decolorized and rendered nearly transparent almost all organs of adult mice as well as the entire body of infant and adult mice. This CUBIC-perfusion protocol enables rapid whole-body and whole-organ imaging at single-cell resolution by using light-sheet fluorescent microscopy. The CUBIC protocol is also applicable to 3D pathology, anatomy, and immunohistochemistry of various organs. These results suggest that whole-body imaging of colorless tissues at high resolution will contribute to organism-level systems biology.

An economical, adaptable and user-friendly drip-perfusion bioreactor system designed for in vitro three dimensional cell culturing

This research project investigated a bioreactor system capable of high density cell growth intended for use in regenerative medicine and protein production. The bioreactor was based on a drip-perfusion concept and constructed with minimal costs, readily available components, and straightforward processes for usage. This study involved the design, construction, and testing of the bioreactor where the results showed promising three dimensional cell growth within a polymer structure. The accessibility of this equipment and the capability of high density, three dimensional cell growth would be suitable for future research in pharmaceutical drug manufacturing, and human organ and tissue regeneration.

Additively manufactured device for dynamic culture of large arrays of 3D tissue engineered constructs

The ability to test large arrays of cell and biomaterial combinations in 3D environments is still rather limited in the context of tissue engineering and regenerative medicine. This limitation can be generally addressed by employing highly automated and reproducible methodologies. This study reports on the development of a highly versatile and upscalable method based on additive manufacturing for the fabrication of arrays of scaffolds, which are enclosed into individualized perfusion chambers. Devices containing eight scaffolds and their corresponding bioreactor chambers are simultaneously fabricated utilizing a dual extrusion additive manufacturing system. To demonstrate the versatility of the concept, the scaffolds, while enclosed into the device, are subsequently surface-coated with a biomimetic calcium phosphate layer by perfusion with simulated body fluid solution. 96 scaffolds are simultaneously seeded and cultured with human osteoblasts under highly controlled bidirectional perfusion dynamic conditions over 4 weeks. Both coated and noncoated resulting scaffolds show homogeneous cell distribution and high cell viability throughout the 4 weeks culture period and CaP-coated scaffolds result in a significantly increased cell number. The methodology developed in this work exemplifies the applicability of additive manufacturing as a tool for further automation of studies in the field of tissue engineering and regenerative medicine.

Intraoperative cerebral perfusion and postoperative cognitive dysfunction in geriatric patients

Background: Inadequate intraoperative cerebral perfusion has been suggested as a possible cause of postoperative cognitive dysfunction (POCD). Methods: We investigated 35 patients aged 65 or older undergoing elective major non-cardiac surgery under standardized general anaesthesia (thiopental, sevoflurane, fentanyl, atracurium). Intraoperative cerebral perfusion was monitored with transcranial Doppler, and near-infrared spectroscopy (NIRS). Arterial blood pressure was monitored continuously with a Finapres device. Mx, an index allowing continuous monitoring of cerebrovascular autoregulation based on the changes in mean arterial blood pressure (MAP) and cerebral blood flow velocity was calculated. Mx >0.5 was defined as disturbed cerebrovascular autoregulation. Cognitive function was measured preoperatively and 7 days postoperatively using the CERAD-NAB Plus test battery. A postoperative decline >1 z-score in at least two of the tested domains was defined as POCD. Data are shown as mean } SD. Results: Mean age was 75 } 7 yrs. Sixteen patients (46%) developed POCD. These patients were older (77 } 8 vs 73 } 7 yrs), had lower MAP (77 } 12 vs 81 } 11 mm Hg), lower cerebral tissue oxygenation indices measured by NIRS (66.8 } 6.0 vs 68.6 } 4.3%) and less efficient cerebrovascular autoregulation (Mx 0.54 } 0.17 and 0.44 } 0.22) than patients without POCD. Disturbed intraoperative cerebrovascular autoregulation was found more often (56 vs 37%) in patients with POCD. However, none of these differences reached statistical significance. Conclusions: Our data show a trend towards subtle changes in intraoperative cerebral perfusion in elderly patients who develop POCD. However, a cause effect relationship must not be assumed and a greater number of patients needs to be investigated patients. However, more patients need to be investigated to confirm and characterize these differences.

Effect of intraluminal distention on microvascular perfusion in the equine small colon

Objective-To determine the effect of experimental intraluminal distention on microvascular perfusion of the small colon in horses.Animals-6 mixed-breed healthy horses (mean age [+/- SD], 9.1 +/- 2 years).Procedure-Under general anesthesia, the small colon was exposed by celiotomy and 3 segments were demarcated. In 1 of these segments, intraluminal obstruction was created by placement of a latex balloon inflated to a pressure of 40 mm Hg (obstructed segment). The other segments were the sham-operated segment and the control segment. Microvascular perfusion was evaluated in the mucosal, submucosal, muscular, and serosal layers by injection of 15-mum-diameter colored microspheres into branches of the caudal mesenteric artery. Recovery of microspheres was performed by tissue digestion, washing, and centrifugation. Distribution of microspheres in the intestinal layers was evaluated by direct observation of stained frozen sections by light microscopy.Results-A significant reduction was observed in total microvascular perfusion of obstructed segments, which was 26.4% of that of control segments. This reduction was not evident in the mucosal layer.Conclusions and Clinical Relevance-Intraluminal distention of the equine small colon wall can promote ischemia by a reduction in microvascular perfusion in the intestinal wall. Intestinal layers do not seem to be affected to the same extent, because the absolute value for mucosal perfusion did not decrease in the obstructed segment.