Cell Density Gradient Research Articles

Free-swimming bacteria transcriptionally respond to shear flow

Surface-attached cells can sense and respond to shear flow, but planktonic (free-swimming) cells are typically assumed to be oblivious to any flow that carries them. Here, we find that planktonic bacteria can transcriptionally respond to flow, inducing expression changes that are beneficial in flow. Specifically, we use microfluidic experiments and quantitative modeling to show that in the presence of flow, planktonic Pseudomonas aeruginosa induce shear rate-dependent genes that promote growth in low-oxygen environments. Untangling this mechanism revealed that in flow, motile P. aeruginosa spatially redistribute, leading to cell density changes that activate quorum sensing, which in turn enhances the oxygen uptake rate. In diffusion-limited environments, including those commonly encountered by bacteria, flow-induced cell density gradients also independently generate oxygen gradients that alter gene expression. Mutants deficient in this flow-responsive mechanism exhibit decreased fitness in flow, suggesting that this dynamic coupling of biological and mechanical processes can be physiologically significant.

Differential proteomics profile of microcapillary networks in response to sound pattern-driven local cell density enhancement

Spatial cell organization and biofabrication of microcapillary networks in vitro has a great potential in tissue engineering and regenerative medicine. This study explores the impact of local cell density enhancement achieved through an innovative sound-based patterning on microcapillary networks formation and their proteomic profile. Human umbilical vein endothelial cells (HUVEC) and human pericytes from placenta (hPC-PL) were mixed in a fibrin suspension. The mild effect of sound-induced hydrodynamic forces condensed cells into architected geometries showing good fidelity to the numerical simulation of the physical process. Local cell density increased significantly within the patterned areas and the capillary-like structures formed following the cell density gradient. Over five days, these patterns were well-maintained, resulting in concentric circles and honeycomb-like structures.Proteomic analysis of the pre-condensed cells cultured for 5 days, revealed over 900 differentially expressed proteins when cells were preassembled through mild-hydrodynamic forces. Gene ontology (GO) enrichment analysis identified cellular components, molecular functions, and biological processes that were up- and down-regulated, providing insights regarding molecular processes influenced by the local density enhancement. Furthermore, we employed Ingenuity Pathway Analysis (IPA) to identify altered pathways and predict upstream regulators. Notably, VEGF-A emerged as one of the most prominent upstream regulators.Accordingly, this study initiates the unraveling of the changes in microcapillary networks at both molecular and proteins level induced by cell condensation obtained through sound patterning. The findings provide valuable insights for further investigation into sound patterning as a biofabrication technique for creating more complex microcapillary networks and advancing in vitro models.

Improved electromagnetic interference shielding performance of silicone rubber/CNTs composites by porous structure design via green supercritical CO2 foaming

In this study, the relationship between porous structure and electromagnetic interference (EMI) shielding performance of polymer foams is established. A green supercritical CO2 foaming technology is utilized to construct silicone rubber (PMVS)/carbon nanotubes (CNTs) foams with tunable porous structures (porosity, cell size, cell density and cell gradient structure). Notably, the introduction of moderate porosity (within 40 %) simultaneously enhanced the total EMI shielding effectiveness (SET) and absorption coefficient (A). PMVS/CNTs foam reached a maximum SET of 62.3 dB, a 35.4 % increase over solid PMVS/CNTs composite. Additionally, small-cell PMVS/CNTs foam shows a higher A under the same porosity, while large-cell foam exhibits higher SET. Moreover, gradient porous PMVS/CNTs, combining advantages of large and small cells, maintains a high A (78 %) and exhibits superior SET (52.7 dB), along with exceptional EMI shielding stability (with SET and A retaining 97.7 % and 99 %, respectively, after 1000 cycles of compression). This work provides a theoretical foundation for optimizing EMI shielding performance by regulating porous structures.

CD8+ Cell Density Gradient across the Tumor Epithelium-Stromal Interface of Non-Muscle Invasive Papillary Urothelial Carcinoma Predicts Recurrence-Free Survival after BCG Immunotherapy.

Bacille Calmette-Guerin (BCG) immunotherapy is the first-line treatment in patients with high-risk non-muscle invasive papillary urothelial carcinoma (NMIPUC), the most common type of bladder cancer. The therapy outcomes are variable and may depend on the immune response within the tumor microenvironment. In our study, we explored the prognostic value of CD8+ cell density gradient indicators across the tumor epithelium-stroma interface of NMIPUC. Clinical and pathologic data were retrospectively collected from 157 NMIPUC patients treated with BCG immunotherapy after transurethral resection. Whole-slide digital image analysis of CD8 immunohistochemistry slides was used for tissue segmentation, CD8+ cell quantification, and the assessment of CD8+ cell densities within the epithelium-stroma interface. Subsequently, the gradient indicators (center of mass and immunodrop) were computed to represent the density gradient across the interface. By univariable analysis of the clinicopathologic factors, including the history of previous NMIPUC, poor tumor differentiation, and pT1 stage, were associated with shorter RFS (p < 0.05). In CD8+ analyses, only the gradient indicators but not the absolute CD8+ densities were predictive for RFS (p < 0.05). The best-performing cross-validated model included previous episodes of NMIPUC (HR = 4.4492, p = 0.0063), poor differentiation (HR = 2.3672, p = 0.0457), and immunodrop (HR = 5.5072, p = 0.0455). We found that gradient indicators of CD8+ cell densities across the tumor epithelium-stroma interface, along with routine clinical and pathology data, improve the prediction of RFS in NMIPUC.

The mechanical regulation of Eph/ephrin signaling in the developing brain.

Eph receptors and their membrane-bound ligands, ephrins, provide key signals in many developmental processes including neuronal guidance. However, despite immense progress in our understanding of Eph/ephrin signaling, discrepancies between in vitro and in vivo work remain. As axon pathfinding is regulated by chemical and mechanical signals, and the mechanical regulation of Eph/ephrin signaling is currently poorly understood, we here investigated the role of mechanical cues in this signaling pathway. Xenopus retinal neurons cultured on soft substrates mechanically resembling brain tissue had the opposite response to ephrinB1 compared to those cultured on glass. Furthermore, in vivo atomic force microscopy data showed that the Xenopus visual area of the brain, the optic tectum, becomes mechanically heterogenous as retinal neurons approach the diencephalon-tectum boundary and begin to innervate it. The stiffness gradient which develops correlates with both a cell density gradient and a concentration gradient of EphB expression detected by hybridization chain reaction. Since EphB/ephrinB signaling in Xenopus retinal neurons is affected by substrate stiffness in vitro, and a stiffness gradient develops across the optic tectum at the time of innervation, our data suggest that mechanical cues could be important in tuning retinotectal mapping through the regulation of chemical signaling. A similar regulation of chemical signaling through tissue mechanics is likely to be important across multiple aspects of neural development, as well as in other organ systems.

Vitamin D induced microbicidal activity against Mycobacterium bovis BCG is dependent on the synergistic activity of bovine peripheral blood cell populations

A growing appreciation is emerging of the beneficial role of vitamin D for health and resistance against infectious diseases, including tuberculosis. However, research has predominantly focused on murine and human species and functional data in bovines is limited. Therefore, the objective of this study was to assess the microbicidal activity and immunoregulatory effect of the vitamin D metabolite 1,25(OH)2D3 on bovine peripheral blood leukocytes (PBL) in response to Mycobacterium bovis BCG (BCG) infection using a combination of functional assays and gene expression profiling. Blood from Holstein-Friesian bull calves with low circulating levels of 25(OH)D was stimulated with 1,25(OH)2D3 for 2 h, and then infected with M. bovis BCG. Results showed that 1,25(OH)2D3 supplementation significantly increased BCG killing by on average 16 %, although responses varied between 1 % and 38 % killing. Serial cell subset depletion was then performed on PBL prior to 1,25(OH)2D3 incubation and BCG infected as before to analyse the contribution of major cell types to mycobacterial growth control. Specific antibodies and either magnetic cell separation or density gradient centrifugation of monocytes, granulocytes, CD3+, CD4+, and CD8+ T lymphocytes were used to capture each cell subset. Results showed that depletion of granulocytes had the greatest impact on BCG growth, leading to a significant enhancement of bacterial colonies. In contrast, depletion of CD4+ or CD8+ T cells individually, or in combination (CD3+), had no impact on mycobacterial growth control. In agreement with our previous data, 1,25(OH)2D3 significantly increased bacterial killing in PBL, in monocyte depleted samples, and a similar trend was observed in the granulocyte depleted subset. In addition, specific analysis of sorted neutrophils treated with 1,25(OH)2D3 showed an enhanced microbicidal activity against both BCG and a virulent strain of M. bovis. Lastly, data showed that 1,25(OH)2D3 stimulation increased reactive oxygen species (ROS) production and the expression of genes encoding host defence peptides (HDP) and pathogen recognition receptors (PRRs), factors that play an important role in the microbicidal activity against mycobacteria. In conclusion, the vitamin D metabolite 1,25(OH)2D3 improves antimycobacterial killing in bovine PBLs via the synergistic activity of monocytes and granulocytes and enhanced activation of innate immunity.

Contribution of parasol-magnocellular pathway ganglion cells to foveal retina in macaque monkey

Parasol-magnocellular pathway ganglion cells form an important output stream of the primate retina and make a major contribution to visual motion detection. They are known to comprise ON and OFF type response polarities but the relative numbers of ON and OFF parasol cells, and the overall contribution of parasol cells to high-acuity foveal vision are not well understood. Here we use antibodies against carbonic anhydrase 8 (CA8) and intracellular injections of the liphilic dye DiI to show that CA8 selectively labels OFF parasol cells in macaque retina. By combined labeling with CA8 antibodies and a previously-described marker for parasol cells (GABAA receptor antibodies), we show that ON and OFF parasol cells each comprise ∼ 6% of all ganglion cells in central retina (each peak density ∼ 3000 cells/mm2 at 5 deg.), and each population comprises ∼ 10% of all ganglion cells in peripheral temporal retina. Thus, the spatial density of parasol cells in central retina is greater than reported by previous anatomical studies, and the central-peripheral gradient in parasol cell density is shallower than previously reported. The data nevertheless predict decline in spatial acuity with visual field eccentricity for both midget-parvocellular pathway and parasol-magnocellular pathway mediated visual functions. The spatial resolving power of the OFF parasol array (peak ∼ 7 cpd) falls short of macaque behavioral grating acuity by at least a factor of three throughout the retina.

A simple integrated microfluidic platform for the research of hydrogels containing gradients in cell density induced breast cancer electrochemotherapy

Cell density is important for tumour metastasis, treatment and prognosis. Characterizing changes in cell density for electrochemotherapy (ECT) can reveal sub-populations in pathological states, and adjust treatment program. In this work, a simple and convenient microfluidic platform was developed to study the effect cell density on ECT by integrating the improved cell gradient generator, cell culture chamber and indium tin oxide interdigital electrodes. Agarose, as extracellular matrix (ECM), was used to 3D cell culture to imitate in vivo microenvironment. The precision and reproducibility of cell density gradient with agarose solution were achieved because the hydrophobic modification of microchannels surface resulted in reducing cell adhesion and residue. ECT cytotoxicity assay with difference in cell densities was studied. The results showed that tumour cell density is one of the most factors for ECT treatment and ECT cytotoxicity has a certain of cell density-depended. But only electroporation on low cell density level, ECM would be one of the most key factors for ECT cytotoxicity, which would provide a new idea for chip-based cell assay in clinical diagnosis and drug screening in ordinary laboratories.

Approaching ideal energy absorption through the multicellular structure with gradient material distribution

Energy absorption of traditional tubular energy absorbers is restricted by the low structure efficiency, which makes the mean crushing force of the structure usually much lower than its yield strength. The combination of a multicellular design and a gradient-thickness strategy significantly improves the energy-absorption ability of thin-walled energy absorbers while reducing weight. Gradient-thickness multicellular tubes (GTMT) were studied in this paper to demonstrate this advantage. Mechanical performances of GTMT with two material-distribution principles, sample gradient thickness (SGT) and modified gradient thickness (MGT), were investigated experimentally and numerically, primarily in terms of folding behaviors, energy-absorption ability, and load-carrying capacity undulation. Wire-cut electrical discharge machining technology was used to create a series of aluminum alloy specimens that were then compressed under quasistatic loading conditions.Following that, finite element method was used to run detailed numerical simulations. The effect of geometric configuration was determined after conducting parametric studies with different cell density and thickness gradient coefficients. The results showed that, compared to a traditional multicellular tube, a gradient-thickness one with MGT material distribution can improve structural efficiency with a stable loading history. Increases in cell density and thickness gradient coefficient have positive effects on energy-absorption ability; however, excessively high parameter values will lead to global bending deformation and weaken the mean crushing force. Therefore, reasonable parameter matching is vital. The result shows that when the cell density reached 9 × 9 and the thickness gradient coefficient reached 1.4, the mean crushing force was 98.16% of the full-plastic strength of the matrix, and there was no irregular deformation, indicating that ideal energy absorption is almost achieved. These achievements pave a way for achieving ideal energy absorption.

Sound-induced morphogenesis of multicellular systems for rapid orchestration of vascular networks

Morphogenesis, a complex process, ubiquitous in developmental biology and many pathologies, is based on self-patterning of cells. Spatial patterns of cells, organoids, or inorganic particles can be forced on demand using acoustic surface standing waves, such as the Faraday waves. This technology allows tuning of parameters (sound frequency, amplitude, chamber shape) under contactless, fast and mild culture conditions, for morphologically relevant tissue generation. We call this method Sound Induced Morphogenesis (SIM). In this work, we use SIM to achieve tight control over patterning of endothelial cells and mesenchymal stem cells densities within a hydrogel, with the endpoint formation of vascular structures. Here, we first parameterize our system to produce enhanced cell density gradients. Second, we allow for vasculogenesis after SIM patterning control and compare our controlled technology against state-of-the-art microfluidic culture systems, the latter characteristic of pure self-organized patterning and uniform initial density. Our sound-induced cell density patterning and subsequent vasculogenesis requires less cells than the microfluidic chamber. We advocate for the use of SIM for rapid, mild, and reproducible morphogenesis induction and further explorations in the regenerative medicine and cell therapy fields.

Engineering slit-like channels for studying the growth of epithelial tissues in 3D-confined spaces.

The development of epithelial lumens in ducts is essential to the functioning of various organs and in organogenesis. Ductal elongation requires the collective migration of cell cohorts in three-dimensional (3D) confined spaces, while maintaining their epithelial integrity. Epithelial lumens generally adopt circular morphologies, however abnormalities in complex physiological environments can lead to the narrowing of glandular spaces that adopt elongated and slit-like morphologies. Here, we describe a simple method to form epithelial tissues in microchannels of various widths (100-300 µm) with a constant height of 25 µm that mimic elongated geometries of glandular spaces. The significance of this biomimetic platform has been evidenced by studying the migration of epithelial cell sheets inside these narrow slits of varying dimensions. We show that the growth of epithelial tissues in 3D-confined slits leads to a gradient of cell density along the slit axis and that the migration cell velocity depends on the extent of the spatial confinement. Our findings indicate that nuclear orientation is higher for leader cells and depends on the slit width, whereas YAP protein was predominantly localized in the nucleus of leader cells. This method will pave the way to studies aiming at understanding how 3D-confined spaces, which are reminiscent of in vivo pathological conditions, can affect the growth and the homeostasis of epithelial tissues.

Cell density-dependent suppression on the development and photosynthetic activities of Sargassum fusiformis embryos by dinoflagellate Karenia mikimotoi

Lots of research has demonstrated that macroalgae can strongly inhibit the growth of harmful algal bloom (HAB) species in general. However, the effects of HABs or HAB-forming species on macrophytes are still largely uncharacterized until now. In the present study, the effects of the dinoflagellate Karenia mikimotoi cell density gradient, live cell suspension (LC), ruptured cell suspension (RC) as well as the cell-free supernatant (FC) of K. mikimotoi at 1000 μg Chla l−1 (~1.0 × 105 cells ml−1) on the development and photosynthesis of Sargassum fusiforme embryos were investigated in a series of laboratory experiments. The results showed that co-cultivation with K. mikimotoi at 500 μg Chla l−1(~5.0 × 104 cells ml−1) and higher cell densities significantly (P<0.05) inhibited the development, pigment content and photosynthetic activities of the embryos. In addition, the inhibitory effects increased with increased cell densities and prolonged exposure time. Compared to the embryos cultured with the F/2 medium (Control), exposure to LC, RC and FC of K. mikimotoi at 1000 μg Chla l−1for 2 weeks all led to decreased relative growth rate (RGR), chlorophyll (Chl) a content, carotenoids (Car) content and photosynthetic activities of the embryos, with LC and RC exhibiting the maximal and the minimal suppression. The dominant inhibitory effects of FC on the embryos indicated that the suppression was mainly caused by the allelochemicals, while the slightest inhibitory effects of RC on the embryos suggested that some intracellular growth-promoting substances were synchronously released when K. mikimotoi cells lyzed. In addition, the most severe growth suppression of embryos by LC indicated that intact cell contact by K. mikimotoi probably also contributed to the inhibitory effects. These results indicated that a dense HAB formed by K. mikimotoi could seriously suppress the development and photosynthesis of S. fusiforme embryos and eventually reduce the seedlings stock.

Guided cellular orientation concurrently with cell density gradient on butterfly wings.

A simple method to create guided cellular orientation is illustrated by assembling fibroblasts on the dorsal side of M. menelaus wings. Moreover, by inserting the wing into tendon fibroblasts suspension at a tilt angle, guided cellular orientation concurrently with the cell density gradient is formed on the butterfly wings.

Magnetic-activated cell sorting (MACS) versus density gradient centrifugation (DGC) for the selection of human sperm in assisted reproductive techniques

Objective: To investigate whether the assisted reproductive techniques (ART) outcomes can be improved by using magnetic-activated cell sorting combined isolate density gradient centrifugation (MACS-DGC), and to compare the results with the conventional isolate density gradient centrifugation (DGC) method. Methods: In the period from January to December 2014, patients who attended the andrology laboratory for sperm DNA fragmentation analysis and underwent the in vitro fertilization and embryo transfer (IVF-ET) treatment were divided into 3 groups, and each group has two subgroups according to the way of fertilization : in vitro fertilization (IVF) as subgroup 1 and intracytoplasmic sperm injection (ICSI) as subgroup 2. Group A (A1, A2 ): DNA fragmentation index(DFI)≥20 and using MACs; Group B (B1, B2): DFI≥20 and using isolate method; Group C (C1, C2): DFI<20 and using isolate method. The fertilization rate, good-quality embryos, implantation rate, pregnancy rate and live-birth rate of each group were analyzed. Results: Similar results were obtained among groups for all the parameters compared: On IVF cycles no significant difference was observed in fertilization rate, percentage of good-quality embryos on day 3, implantation rate, positive beta-hCG tests, and live-birth rate among the three subgroups (A1 vs B1 vs C1). Between the subgroups of ICSI cycles (A2 vs B2 vs C2) the percentage of high-quality embryos, pregnancy rate, implantation rate and live-birth rate were all significantly higher in the MACS-DGC (A2) group compared to DGC alone (B2). Conclusion: Based on the results, MACS as a part of sperm preparation technique will not improve sperm fertilizing potential, embryos quality, implantation rate, pregnancy rate or live-birth rate of IVF, but it may help select the most fertile sperm and to improve clinical outcomes of ICSI.

An optimized protocol for the preparation of oxygen-evolving thylakoid membranes from Cyclotella meneghiniana provides a tool for the investigation of diatom plastidic electron transport

BackgroundThe preparation of functional thylakoid membranes from diatoms with a silica cell wall is still a largely unsolved challenge. Therefore, an optimized protocol for the isolation of oxygen evolving thylakoid membranes of the centric diatom Cyclotella meneghiniana has been developed. The buffer used for the disruption of the cells was supplemented with polyethylene glycol based on its stabilizing effect on plastidic membranes. Disruption of the silica cell walls was performed in a French Pressure cell and subsequent linear sorbitol density gradient centrifugation was used to isolate the thylakoid membrane fraction.ResultsSpectroscopic characterization of the thylakoids by absorption and 77 K fluorescence spectroscopy showed that the photosynthetic pigment protein complexes in the isolated thylakoid membranes were intact. This was supported by oxygen evolution measurements which demonstrated high electron transport rates in the presence of the artificial electron acceptor DCQB. High photosynthetic activity of photosystem II was corroborated by the results of fast fluorescence induction measurements. In addition to PSII and linear electron transport, indications for a chlororespiratory electron transport were observed in the isolated thylakoid membranes. Photosynthetic electron transport also resulted in the establishment of a proton gradient as evidenced by the quenching of 9-amino-acridine fluorescence. Because of their ability to build-up a light-driven proton gradient, de-epoxidation of diadinoxanthin to diatoxanthin and diatoxanthin-dependent non-photochemical quenching of chlorophyll fluorescence could be observed for the first time in isolated thylakoid membranes of diatoms. However, the ∆pH, diadinoxanthin de-epoxidation and diatoxanthin-dependent NPQ were weak compared to intact diatom cells or isolated thylakoids of higher plants.ConclusionsThe present protocol resulted in thylakoids with a high electron transport capacity. These thylakoids can thus be used for experiments addressing various aspects of the photosynthetic electron transport by, e.g., employing artificial electron donors and acceptors which do not penetrate the diatom cell wall. In addition, the present isolation protocol yields diatom thylakoids with the potential for xanthophyll cycle and non-photochemical quenching measurements. However, the preparation has to be further refined before these important topics can be addressed systematically.

Oxygen-dependent generation of a graded polydopamine coating on nanofibrous materials for controlling stem cell functions.

Substrates modified with gradient surface chemistry are of fundamental importance for designing a new bio-interface in biomaterial research and tissue engineering. However, current gradient fabrication strategies are not easily accessible to most laboratories due to complex, expensive, and expertise-requiring procedures. In this study, we generated a gradient of polydopamine (PD) coating on a PLLA nanofiber surface using a spatially restricted supply of oxygen in the reaction solution. Analysis of the oxygen distribution revealed that oxygen availability varied along different reaction solution depths during dopamine polymerization. We then extensively investigated the effects of different parameters, such as tilting angle, reaction time, pH of the reaction solution, and concentration of dopamine, on PD gradient formation, which should be appropriately modulated for PD gradient on nanofibers. Further, culturing of human mesenchymal stem cells (hMSCs) on the PD gradient nanofiber resulted in a gradient of adhesion and spreading from high to low PD coating. However, the proliferation rate was not affected by the PD gradient, with an approximately 3-fold change after 5 days of culture. Maintenance of the stem cell density gradient on the PD gradient nanofiber resulted in controlled osteogenic differentiation, which was greater in the higher PD-coated area. Interestingly, stemness analysis showed a reverse trend relative to osteogenic differentiation of hMSCs. In summary, the spatially controlled polymerization of dopamine can be a versatile tool to generate substrates with gradient surface chemistry, which holds promise to direct stem cell behavior.

Preparation of polymer foams with a gradient of cell size: Further exploring the nucleation effect of porous inorganic materials in polymer foaming

Polymeric foams with gradient of cell size have shown significant potential in many applications, but, the fabrication of such foams is difficult. By producing blowing agent concentration or temperature gradient inside the sample, some recently developed methods have successfully prepared gradient polymer foams, whereas, precise control system and/or delicate operation are highly needed. Herein, we reported a simple method to obtain cell size gradient in polymer foams by a batch foaming of the polymers on a substrate with nanopore structure. Anodized aluminum oxide (AAO) film modified with a fluorinated silane was used as the substrate. Polymers including polystyrene, polymethyl methacrylate, and polylactic acid were compressed onto the modified AAO film, and then foamed by a batch foaming process using scCO2 as blowing agent. SEM characterizations on the foam morphology revealed that for all the samples, the size of cell near the substrate was smaller and gradually increased along the distance from AAO surface. It is also found that the gradient structure varied with the polymer type. Specifically, polylactic acid foams showed a perfect gradient of cell size from ∼13μm to ∼40μm in a thickness of more than 1.0mm, providing significant potentials in many applications. Moreover, by choosing the substrates with nanopores of different size and/or changing the foaming condition, the foam morphologies including cell size, cell density, and cell size gradient range can be readily tuned.

Engineering zonal cartilage through bioprinting collagen type II hydrogel constructs with biomimetic chondrocyte density gradient.

BackgroundCartilage tissue engineering is a promising approach for repairing and regenerating cartilage tissue. To date, attempts have been made to construct zonal cartilage that mimics the cartilaginous matrix in different zones. However, little attention has been paid to the chondrocyte density gradient within the articular cartilage. We hypothesized that the chondrocyte density gradient plays an important role in forming the zonal distribution of extracellular matrix (ECM).MethodsIn this study, collagen type II hydrogel/chondrocyte constructs were fabricated using a bioprinter. Three groups were created according to the total cell seeding density in collagen type II pre-gel: Group A, 2 × 107 cells/mL; Group B, 1 × 107 cells/mL; and Group C, 0.5 × 107 cells/mL. Each group included two types of construct: one with a biomimetic chondrocyte density gradient and the other with a single cell density. The constructs were cultured in vitro and harvested at 0, 1, 2, and 3 weeks for cell viability testing, reverse-transcription quantitative PCR (RT-qPCR), biochemical assays, and histological analysis.ResultsWe found that total ECM production was positively correlated with the total cell density in the early culture stage, that the cell density gradient distribution resulted in a gradient distribution of ECM, and that the chondrocytes’ biosynthetic ability was affected by both the total cell density and the cell distribution pattern.ConclusionsOur results suggested that zonal engineered cartilage could be fabricated by bioprinting collagen type II hydrogel constructs with a biomimetic cell density gradient. Both the total cell density and the cell distribution pattern should be optimized to achieve synergistic biological effects.

A 3-dimensional DTI MRI-based model of GBM growth and response to radiation therapy.

Glioblastoma (GBM) is both the most common and the most aggressive intra-axial brain tumor, with a notoriously poor prognosis. To improve this prognosis, it is necessary to understand the dynamics of GBM growth, response to treatment and recurrence. The present study presents a mathematical diffusion-proliferation model of GBM growth and response to radiation therapy based on diffusion tensor (DTI) MRI imaging. This represents an important advance because it allows 3-dimensional tumor modeling in the anatomical context of the brain. Specifically, tumor infiltration is guided by the direction of the white matter tracts along which glioma cells infiltrate. This provides the potential to model different tumor growth patterns based on location within the brain, and to simulate the tumor's response to different radiation therapy regimens. Tumor infiltration across the corpus callosum is simulated in biologically accurate time frames. The response to radiation therapy, including changes in cell density gradients and how these compare across different radiation fractionation protocols, can be rendered. Also, the model can estimate the amount of subthreshold tumor which has extended beyond the visible MR imaging margins. When combined with the ability of being able to estimate the biological parameters of invasiveness and proliferation of a particular GBM from serial MRI scans, it is shown that the model has potential to simulate realistic tumor growth, response and recurrence patterns in individual patients. To the best of our knowledge, this is the first presentation of a DTI-based GBM growth and radiation therapy treatment model.

Identification of the Centrifuged Lipoaspirate Fractions Suitable for Postgrafting Survival.

The Coleman centrifugation procedure generates fractions with different adipocyte and progenitor cell densities. This study aimed to identify all fractions that are feasible for implantation. Human lipoaspirates were processed by Coleman centrifugation. The centrifugates were divided arbitrarily into upper, middle, and lower layers. Adipocyte viability, morphology, numbers of stromal vascular fraction cells, and adipose-derived mesenchymal stem cells of each layer were determined. The 12-week volume retention of subcutaneously implanted 0.3-ml lipoasperate of each layer was investigated in an athymic mice model. Most damaged adipocytes were located in the upper layers, whereas the intact adipocytes were distributed in the middle and lower layers. A gradient of stromal vascular fraction cell density was formed in the centrifugates. The implant volume retentions of samples from the upper, middle, and lower layers were 33.44 ± 5.9, 55.11 ± 4.4, and 71.2 ± 5.8 percent, respectively. Furthermore, the middle and lower layers contained significantly more adipose-derived stem cells than did the upper layer. The lower layer contains more viable adipocytes and stromal vascular fraction cells leading to the highest implant volume retention, whereas the most impaired cells are distributed in the upper layer, leading to the least volume retention. Although with a lower stromal vascular fraction content, the middle layer has a substantial number of intact adipocytes that are capable of retaining partial adipose tissue volume after implantation, suggesting that the middle layer may be an alternative fat source when large volumes of fat grafts are needed for transplantation.