Synthetic and living micropropellers for convection-enhanced nanoparticle transport.

Nanoparticles (NPs) have emerged as an advantageous drug delivery platform for the treatment of various ailments including cancer and cardiovascular and inflammatory diseases. However, their efficacy in shuttling materials to diseased tissue is hampered by a number of physiological barriers. One hurdle is transport out of the blood vessels, compounded by difficulties in subsequent penetration into the target tissue. Here, we report the use of two distinct micropropellers powered by rotating magnetic fields to increase diffusion-limited NP transport by enhancing local fluid convection. In the first approach, we used a single synthetic magnetic microrobot called an artificial bacterial flagellum (ABF), and in the second approach, we used swarms of magnetotactic bacteria (MTB) to create a directable "living ferrofluid" by exploiting ferrohydrodynamics. Both approaches enhance NP transport in a microfluidic model of blood extravasation and tissue penetration that consists of microchannels bordered by a collagen matrix.

Extracellular matrix microarrays to study inductive signaling for endoderm specification.

During tissue development, stem and progenitor cells are faced with fate decisions coordinated by microenvironmental cues. Although insights have been gained from in vitro and in vivo studies, the role of the microenvironment remains poorly understood due to the inability to systematically explore combinations of stimuli at a large scale. To overcome such restrictions, we implemented an extracellular matrix (ECM) array platform that facilitates the study of 741 distinct combinations of 38 different ECM components in a systematic, unbiased and high-throughput manner. Using embryonic stem cells as a model system, we derived definitive endoderm progenitors and applied them to the array platform to study the influence of ECM, including the interactions of ECM with growth factor signaling, on the specification of definitive endoderm cells towards the liver and pancreas fates. We identified ECM combinations that influence endoderm fate decisions towards these lineages, and demonstrated the utility of this platform for studying ECM-mediated modifications to signal activation during liver specification. In particular, defined combinations of fibronectin and laminin isoforms, as well as combinations of distinct collagen subtypes, were shown to influence SMAD pathway activation and the degree of hepatic differentiation. Overall, our systematic high-throughput approach suggests that ECM components of the microenvironment have modulatory effects on endoderm differentiation, including effects on lineage fate choice and cell adhesion and survival during the differentiation process. This platform represents a robust tool for analyzing effects of ECM composition towards the continued improvement of stem cell differentiation protocols and further elucidation of tissue development processes. STATEMENT OF SIGNIFICANCE: Cellular microarrays can provide the capability to perform high-throughput investigations into the role of microenvironmental signals in a variety of cell functions. This study demonstrates the utility of a high-throughput cellular microarray approach for analyzing the effects of extracellular matrix (ECM) in liver and pancreas differentiation of endoderm progenitor cells. Despite an appreciation that ECM is likely involved in these processes, the influence of ECM, particularly combinations of matrix proteins, had not been systematically explored. In addition to the identification of relevant ECM compositions, this study illustrates the capability of the cellular microarray platform to be integrated with a diverse range of cell fate measurements, which could be broadly applied towards the investigation of cell fate regulation in other tissue development and disease contexts.

Pluripotent stem cell-derived hepatocyte-like cells.

Liver disease is an important clinical problem, impacting over 30 million Americans and over 600 million people worldwide. It is the 12th leading cause of death in the United States and the 16th worldwide. Due to a paucity of donor organs, several thousand Americans die yearly while waiting for liver transplantation. Unfortunately, alternative tissue sources such as fetal hepatocytes and hepatic cell lines are unreliable, difficult to reproduce, and do not fully recapitulate hepatocyte phenotype and functions. As a consequence, alternative cell sources that do not have these limitations have been sought. Human embryonic stem (hES) cell- and induced pluripotent stem (iPS) cell-derived hepatocyte-like cells may enable cell based therapeutics, the study of the mechanisms of human disease and human development, and provide a platform for screening the efficacy and toxicity of pharmaceuticals. iPS cells can be differentiated in a step-wise fashion with high efficiency and reproducibility into hepatocyte-like cells that exhibit morphologic and phenotypic characteristics of hepatocytes. In addition, iPS-derived hepatocyte-like cells (iHLCs) possess some functional hepatic activity as they secrete urea, alpha-1-antitrypsin, and albumin. However, the combined phenotypic and functional traits exhibited by iHLCs resemble a relatively immature hepatic phenotype that more closely resembles that of fetal hepatocytes rather than adult hepatocytes. Specifically, iHLCs express fetal markers such as alpha-fetoprotein and lack key mature hepatocyte functions, as reflected by drastically reduced activity (~0.1%) of important detoxification enzymes (i.e. CYP2A6, CYP3A4). These key differences between iHLCs and primary adult human hepatocytes have limited the use of stem cells as a renewable source of functional adult hepatocytes for in vitro and in vivo applications. Unfortunately, the developmental pathways that control hepatocyte maturation from a fetal into an adult hepatocyte are poorly understood, which has hampered the field in its efforts to induce further maturation of iPS-derived hepatic lineage cells. This review analyzes recent developments in the derivation of hepatocyte-like cells, and proposes important points to consider and assays to perform during their characterization. In the future, we envision that iHLCs will be used as in vitro models of human disease, and in the longer term, provide an alternative cell source for drug testing and clinical therapy.

InVERT molding for scalable control of tissue microarchitecture.

Complex tissues contain multiple cell types that are hierarchically organized within morphologically and functionally distinct compartments. Construction of engineered tissues with optimized tissue architecture has been limited by tissue fabrication techniques, which do not enable versatile microscale organization of multiple cell types in tissues of size adequate for physiological studies and tissue therapies. Here we present an 'Intaglio-Void/Embed-Relief Topographic molding' method for microscale organization of many cell types, including induced pluripotent stem cell-derived progeny, within a variety of synthetic and natural extracellular matrices and across tissues of sizes appropriate for in vitro, pre-clinical, and clinical studies. We demonstrate that compartmental placement of non-parenchymal cells relative to primary or induced pluripotent stem cell-derived hepatocytes, compartment microstructure, and cellular composition modulate hepatic functions. Configurations found to sustain physiological function in vitro also result in survival and function in mice for at least 4 weeks, demonstrating the importance of architectural optimization before implantation.

Review of an in vitro microfluidic model of sickle cell vaso-occlusion.

Vaso-occlusive events are responsible for the majority of morbidity and mortality in sickle cell disease. Predisposing conditions are unclear, and proximal causes have not been established. Despite decades of intense study, until recently there has not been a well-controlled in vitro model of sickle cell vaso-occlusion. We have reported the development and initial use of such a model. Our experimental device relies on microfluidic technology and has allowed the initial analysis of the in vitro process of vaso-occlusion in terms of control parameters such as driving pressure, local oxygen concentration and flow vessel size. Our work demonstrates the potential of this type of device to lead to greater understanding of vaso-occlusive pathology including the role of adhesion molecules and inflammatory factors and possibly to improvements in monitoring and searches for new treatments.

Sickle cell vasoocclusion and rescue in a microfluidic device.

The pathophysiology of sickle cell disease is complicated by the multiscale processes that link the molecular genotype to the organismal phenotype: hemoglobin polymerization occurring in milliseconds, microscopic cellular sickling in a few seconds or less [Eaton WA, Hofrichter J (1990) Adv Protein Chem 40:63-279], and macroscopic vessel occlusion over a time scale of minutes, the last of which is necessary for a crisis [Bunn HF (1997) N Engl J Med 337:762-769]. Using a minimal but robust artificial microfluidic environment, we show that it is possible to evoke, control, and inhibit the collective vasoocclusive or jamming event in sickle cell disease. We use a combination of geometric, physical, chemical, and biological means to quantify the phase space for the onset of a jamming event, as well as its dissolution, and find that oxygen-dependent sickle hemoglobin polymerization and melting alone are sufficient to recreate jamming and rescue. We further show that a key source of the heterogeneity in occlusion arises from the slow collective jamming of a confined, flowing suspension of soft cells that change their morphology and rheology relatively quickly. Finally, we quantify and investigate the effects of small-molecule inhibitors of polymerization and therapeutic red blood cell exchange on this dynamical process. Our experimental study integrates the dynamics of collective processes associated with occlusion at the molecular, polymer, cellular, and tissue level; lays the foundation for a quantitative understanding of the rate-limiting processes; and provides a potential tool for optimizing and individualizing treatment, and identifying new therapies.

Effect of cell-cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells.

Heterotypic cell interaction between parenchymal cells and nonparenchymal neighbors has been reported to modulate cell growth, migration, and/or differentiation. In both the developing and adult liver, cell-cell interactions are imperative for coordinated organ function. In vitro, cocultivation of hepatocytes and nonparenchymal cells has been used to preserve and modulate the hepatocyte phenotype. We summarize previous studies in this area as well as recent advances in microfabrication that have allowed for more precise control over cell-cell interactions through 'cellular patterning' or 'micropatterning'. Although the precise mechanisms by which nonparenchymal cells modulate the hepatocyte phenotype remain unelucidated, some new insights on the modes of cell signaling, the extent of cell-cell interaction, and the ratio of cell populations are noted. Proposed clinical applications of hepatocyte cocultures, typically extracorporeal bioartificial liver support systems, are reviewed in the context of these new findings. Continued advances in microfabrication and cell culture will allow further study of the role of cell communication in physiological and pathophysiological processes as well as in the development of functional tissue constructs for medical applications.

Prenatal detection and mapping of a distal 8p deletion associated with congenital heart disease.

We report the prenatal diagnosis, at 18 weeks' gestational age of a del(8)(p23.1-->pter) in a fetus with an atrio-ventricular canal, persistent left superior vena cava and hypoplastic right ventricle detected by sonographic imaging. We further refine the breakpoints associated with this defect using fluorescent in situ hybridization analysis (FISH). Our findings correlate with recent reports of the localization and importance of GATA4 (a zinc finger transcription factor) in cardiac development. Though microcephaly, mental retardation and typical behavioural features are well described in various deletions in 8p, the absence of notable microcephaly in this case raises the possibility for a separate genetic aetiology for some of these features. Indeed, primary autosomal recessive microcephaly (MCPH1) was recently mapped to a nearby region and may be the cause for this frequent observation in some cases of 8p deletions. These observations illustrate the role of FISH in prenatal diagnosis and refinement of chromosomal breakpoints. In addition, mappings of loci significant for cardiac development are presented. Our findings suggest that some features of the 8p deletion syndrome may ultimately be uncoupled from one another, and underscore the need for further study of this region of chromosome 8, in order to achieve adequate information for genetic counselling.

Numerical model of fluid flow and oxygen transport in a radial-flow microchannel containing hepatocytes.

The incorporation of monolayers of cultured hepatocytes into an extracorporeal perfusion system has become a promising approach for the development of a temporary bioartificial liver (BAL) support system. In this paper we present a numerical investigation of the oxygen tension, shear stress, and pressure drop in a bioreactor for a BAL composed of plasma-perfused chambers containing monolayers of porcine hepatocytes. The chambers consist of microfabricated parallel disks with center-to-edge radial flow. The oxygen uptake rate (OUR), measured in vitro for porcine hepatocytes, was curve-fitted using Michaelis-Menten kinetics for simulation of the oxygen concentration profile. The effect of different parameters that may influence the oxygen transport inside the chambers, such as the plasma flow rate, the chamber height, the initial oxygen tension in the perfused plasma, the OUR, and K(m) was investigated. We found that both the plasma flow rate and the initial oxygen tension may have an important effect upon oxygen transport. Increasing the flow rate and/or the inlet oxygen tension resulted in improved oxygen transport to cells in the radial-flow microchannels, and allowed significantly greater diameter reactor without oxygen limitation to the hepatocytes. In the range investigated in this paper (10 microns < H < 100 microns), and for a constant plasma flow rate, the chamber height, H, had a negligible effect on the oxygen transport to hepatocytes. On the contrary, it strongly affected the mechanical stress on the cells that is also crucial for the successful design of the BAL reactors. A twofold decrease in chamber height from 50 to 25 microns produced approximately a fivefold increase in maximal shear stress at the inlet of the reactor from 2 to 10 dyn/cm2. Further decrease in chamber height resulted in shear stress values that are physiologically unrealistic. Therefore, the channel height needs to be carefully chosen in a BAL design to avoid deleterious hydrodynamic effects on hepatocytes.

Oxygen consumption characteristics of porcine hepatocytes.

Oxygen uptake rate (OUR) of hepatocytes is an important parameter for the design of bioartificial liver assist (BAL) devices. Porcine hepatocytes were cultured in a specially constructed measurement chamber with an incorporated mixing system and a Clark polarographic oxygen electrode. Signal noise associated with conventional Clark electrode implementations was circumvented by the combination of real time digital numerical averaging and subsequent finite impulse response (FIR) spectral filtering. Additional software allowed for the automated generation of cellular oxygen consumption coefficients, namely, Vmax and K0.5, adding a high degree of objectivity to parameter determination. Optimization of the above numerical techniques identified a 0.1 Hz/200 data point sample size and a 0.004 Hz cutoff frequency as ideal parameters. Vmax values obtained for porcine hepatocytes during the first two weeks of culture showed a maximal consumption of 0.9 nmole/sec/10(6) cells occurring on Day 4 post seeding, and a gradual decrease to 0.31 nmole/sec/10(6) cells by Day 15. K0.5 values increased from 2 mm Hg on Day 2 to 8 mm Hg by Day 8, with gradual subsequent decrease to 4 mm Hg by Day 15. The Vmax and K0.5 values measured for porcine cells were higher than maximal values for rat hepatocytes (Vmax: 0.43 nmole/sec/10(6) cells, K0.5: 5.6 mmHg) and thus may necessitate significantly altered BAL device design conditions to ensure no oxygen limitations. Finally, these results highlight the need for species specific characterization of cellular function for optimal BAL device implementations.

Micropatterning cells in tissue engineering.

Recent advances in tissue engineering have been facilitated by the ability to control the environment of cells in vitro. Modulation of cell-extracellular matrix, cell-substrate, and cell-cytokine interactions have proven to be useful in organotypic cultures of many kinds. In some cases, such as skin, bone marrow, and liver, the recovery of physiologic tissue function is enhanced by co-cultivation of two or more cell types together. The in vitro dependence of tissue function on cell-cell interactions is reminiscent of cellular cues during embryogenesis, or adult interactions between parenchymal cells and supporting stroma.

Probing heterotypic cell interactions: hepatocyte function in microfabricated co-cultures.

Replacement of liver function using extracorporeal bioartificial systems has been attempted with limited success. The instability of the hepatocyte phenotype in vitro has restricted the useful lifetime of these devices. Co-cultivation of hepatocytes with mesenchymal cells is one method that has been widely utilized to stabilize the liver-specific function of isolated cells; however, co-culture has yet to be successfully incorporated in a bioreactor setting. In this study, we probed heterotypic cell interactions in co-cultures of hepatocytes and 3T3 in order to better understand the cellular microenvironment necessary to induce and stabilize liver-specific functions. Using microfabrication and conventional techniques to control the heterotypic interface, the effects of varying degrees of heterotypic interaction on tissue function (albumin and urea synthesis) were examined. Our data indicated maximal induction of liver-specific functions in cultures with maximal initial heterotypic interaction, and that induction of hepatic functions in hepatocytes was increased in the vicinity of fibroblasts as compared to hepatocytes far from the heterotypic interface. Furthermore, our data suggested that heterotypic cell contact is necessary for induction of these functions. These studies will aid in the formation of design criteria for a co-culture based bioartificial liver, as well as provide a useful tool to study the role of heterotypic and homotypic interactions in liver physiology and pathophysiology.

Microfabrication of hepatocyte/fibroblast co-cultures: role of homotypic cell interactions.

Cell-cell interactions are important in embryogenesis, in adult physiology and pathophysiology of many disease processes. Co-cultivation of parenchymal and mesenchymal cells has been widely utilized as a paradigm for the study of cell-cell interactions in vitro. In addition, co-cultures of two cell types provide highly functional tissue constructs for use in therapeutic or investigational applications. The inherent complexity of such co-cultures creates difficulty in characterization of cell-cell interactions and their effects on function. In the present study, we utilize conventional "randomly distributed" co-cultures of primary rat hepatocytes and murine 3T3-J2 fibroblasts to investigate the role of increasing fibroblast density on hepatic function. In addition, we utilize microfabrication techniques to localize both cell populations in patterned configurations on rigid substrates. This technique allowed the isolation of fibroblast number as an independent variable in hepatic function. Notably, homotypic hepatocyte interactions were held constant by utilization of similar hepatocyte patterns in all conditions, and the heterotypic interface (region of contact between cell populations) was also held constant. Co-cultures were probed for synthetic and metabolic markers of liver-specific function. The data suggest that fibroblast number plays a role in modulation of hepatocellular response through homotypic fibroblast interactions. The response to changes in fibroblast number are distinct from those attributed to increased contact between hepatocytes and fibroblasts. This approach will allow further elucidation of the complex interplay between two cell types as they form a functional model tissue in vitro or as they interact in vivo to form a functional organ.

Role of microliths in the aetiology of chronic submandibular sialadenitis: a clinicopathological investigation of 154 cases.

AIMS: Confusion about the aetiology and pathogenesis of chronic submandibular sialadenitis led to the present investigation of 154 cases in which many clinical and histological features were analysed. METHODS AND RESULTS: By far the greatest number of histological factors, namely liths, atrophy, fibrosis, parenchymal inflammation, lymphoid germinal centres, mucous and ciliary metaplasia, salivary extravasation and glycosaminoglycan accumulation, was related to the degree of inflammation, which appears to be of the greatest importance in the aetiology and pathogenesis. Inflammation, atrophy and fibrosis were related to duration of symptoms, which supports the concept of a chronological progression through increasingly severe histological changes. CONCLUSIONS: Inflammation possibly arises from ascending infection in a normal gland and exerts an obstructive and destructive effect on the parenchyma with the development of the related histological changes and a vicious circle involving further ascending infection. Normal glands contain microliths that possibly by localized obstruction cause atrophic foci that are reservoirs for ascending infection. Microliths and liths were unrelated: microliths were related to age as in normal glands whereas liths were related to duration of symptoms and appeared to be secondary to the sialadenitis. Many glands showed minimal changes, which raises the possibility of conservative treatment.

Controlling cell interactions by micropatterning in co-cultures: hepatocytes and 3T3 fibroblasts.

The repair or replacement of damaged tissues using in vitro strategies has focused on manipulation of the cell environment by modulation of cell-extracellular matrix interactions, cell-cell interactions, or soluble stimuli. Many of these environmental influences are easily controlled using macroscopic techniques; however, in co-culture systems with two or more cell types, cell-cell interactions have been difficult to manipulate precisely using similar methods. Although microfabrication has been widely utilized for the spatial control of cells in culture, these methods have never been adapted to the simultaneous co-cultivation of more than one cell type. We have developed a versatile technique for micropatterning of two different cell types based on existing strategies for surface modification with aminosilanes linked to biomolecules and the manipulation of serum content of cell culture media. This co-culture technique allowed manipulation of the initial cellular microenvironment without variation of cell number. Specifically, we were able to control the level of homotypic interaction in cultures of a single cell type and the degree of heterotypic contact in co-cultures over a wide range. This methodology has potential applications in tissue engineering, implant biology, and developmental biology, both in the arena of basic science and optimization of function for technological applications.

A comparison of nasal resistance in white Caucasians and blacks.

Total nasal resistance was measured in 42 white caucasian and 32 black healthy adults by both anterior and posterior rhinomanometry after using a nasal decongestant. The nasal airway resistance was found to be lower in the blacks compared to the caucasians by both anterior and posterior methods. The mean total nasal airway resistance was 0.136 Pa/ cm3/s in the blacks and 0.179 Pa/cm3/s in the caucasians by the anterior method, and 0.134 Pa/cm3/s and 0.161 Pa/cm3/s, respectively, by the posterior method. These differences were highly statistically significant.

Selective adhesion of hepatocytes on patterned surfaces.

Successful development of cell-biased bioartificial liver devices necessitates the establishment of techniques and designs for long-term, stable hepatocellular function and efficient transport of nutrients and wastes within the device. Given the relatively large cell mass that one must consider, one possible solution involves the use of micropatterning technology to sandwich hepatocytes aligned in rows between two micropatterned surfaces. Rows of cells would alternate with hepatocyte-free areas, creating efficient transport channels for fluid flow and nutrient exchange. Ultimately, this type of device could also be used as a three-dimensional construct for investigating a variety of cell-surface, cell-extracellular matrix, and cell-cell interactions. To achieve this goal, one must develop techniques for selectively adhering hepatocytes to solid substrates. In this study, reproducible, selective adhesion of hepatocytes on a glass substrate with large regions of adhesive (AS) and nonadhesive (NAS) surfaces was obtained. The AS had hydrophilic characteristics, enhancing deposition of collagen molecules from an aqueous solution, and subsequent hepatocyte adhesion, whereas the NAS had hydrophobic properties and remained collagen-free and hepatocyte-free. In addition, a reproducible processing technique for obtaining patterns of hepatocytes was developed and optimized, using a surface with a single AS band as a first approximation to a micropatterned device. This was achieved by spincoating an aqueous collagen type I solution (0.1 mg/mL) on a banded surface at 500 rpm for 25 seconds. The morphology and long-term function of the hepatocytes attached to AS in nonbanded and banded surface configurations was assessed by mimicking sandwich culture and was shown to be similar to stable, differentiated sandwich cultures. Mathematical modeling was used to determine critical design criteria for the hypothetical micropatterned device. The oxygen distribution and viscous pressure drop were modeled along a typical microchannel and limited to in vivo values. An optimal channel length of 0.6 cm and a flow rate of 2.0 x 10(-6) mL/s were obtained for a channel of 100 microns in width and 10 microns in height. These values were reasonable in terms of practical implementation.

Evaluation of a CCD-based facial measurement system.

This paper describes the configuration and evaluation of a CCD-based biostereometric system at the Department of Orthodontics, Kings College Dental Hospital, London. The system has been designed to allow rapid and accurate measurement of facial soft-tissue shape with a minimum of traditional photogrammetric training. This has been achieved by automatic camera calibration and stereo matching, performed on a Sun Microsystem 3/160 workstation. In summary, the characteristics of the system are: (a) non-contract three-dimensional (3D) measurement of soft-tissue; (b) de-skilled, requiring little or no photogrammetric training; (c) rapid data acquisition, combined with slower off-line processing; (d) processing of resulting 3D surface information to yield cross-sections; (e) surface measurement accurate to within 0.5 mm. Accuracy assessment was performed using a manual electromechanical non-contact measurement device with precision exceeding the requirement of the biostereometric system.

Tooth positions in the natural and complete artificial dentitions, with special reference to the incisor teeth: an interactive on-line computer analysis.

A comparison of tooth positions and soft tissue profiles was made between samples of edentulous patients recently provided with new complete dentures, and dentate subjects. The dentures were marked with lead foil on the most prominent incisor teeth and labial surfaces of the flanges. Lateral skull radiographs were made with the jaws in centric relation. The dentate subjects were recorded with the teeth held in centric occlusion. Discrete soft and hard tissue landmarks were identified and recorded using an interactive on-line computer system, enabling linear and angular measurements to be made. A mathematical comparison was produced employing a series of independent t-tests. Composite computer tracings were also produced. Important differences were found between the two groups, in which the influence of the upper lip length, mandibular resorption, and selected overbite were identified as factors determining artificial tooth position.