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At least 289 records · Page 16

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NASA NTRS1996

Identification of the cAMP response element that controls transcriptional activation of the insulin-like growth factor-I gene by prostaglandin E2 in osteoblasts

Insulin-like growth factor-I (IGF-I), a multifunctional growth factor, plays a key role in skeletal growth and can enhance bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other agents that increase cAMP activated IGF-I gene transcription in primary rat osteoblast cultures through promoter 1 (P1), the major IGF-I promoter, and found that transcriptional induction was mediated by protein kinase A. We now have identified a short segment of P1 that is essential for full hormonal regulation and have characterized inducible DNA-protein interactions involving this site. Transient transfections of IGF-I P1 reporter genes into primary rat osteoblasts showed that the 328-base pair untranslated region of exon 1 was required for a full 5.3-fold response to PGE2; mutation in a previously footprinted site, HS3D (base pairs +193 to +215), reduced induction by 65%. PGE2 stimulated nuclear protein binding to HS3D. Binding, as determined by gel mobility shift assay, was not seen in nuclear extracts from untreated osteoblast cultures, was detected within 2 h of PGE2 treatment, and was maximal by 4 h. This DNA-protein interaction was not observed in cytoplasmic extracts from PGE2-treated cultures, indicating nuclear localization of the protein kinase A-activated factor(s). Activation of this factor was not blocked by cycloheximide (Chx), and Chx did not impair stimulation of IGF-I gene expression by PGE2. In contrast, binding to a consensus cAMP response element (CRE; 5'-TGACGTCA-3') from the rat somatostatin gene was not modulated by PGE2 or Chx. Competition gel mobility shift analysis using mutated DNA probes identified 5'-CGCAATCG-3' as the minimal sequence needed for inducible binding. All modified IGF-I P1 promoterreporter genes with mutations within this CRE sequence also showed a diminished functional response to PGE2. These results identify the CRE within the 5'-untranslated region of IGF-I exon 1 that is required for hormonal activation of IGF-I gene transcription by cAMP in osteoblasts.

NASA Discipline Musculoskeletal↗

NASA NTRS1996

Immunohistochemical evidence of rapid extracellular matrix remodeling after iron-particle irradiation of mouse mammary gland

High-LET radiation has unique physical and biological properties compared to sparsely ionizing radiation. Recent studies demonstrate that sparsely ionizing radiation rapidly alters the pattern of extracellular matrix expression in several tissues, but little is known about the effect of heavy-ion radiation. This study investigates densely ionizing radiation-induced changes in extracellular matrix localization in the mammary glands of adult female BALB/c mice after whole-body irradiation with 0.8 Gy 600 MeV iron particles. The basement membrane and interstitial extracellular matrix proteins of the mammary gland stroma were mapped with respect to time postirradiation using immunofluorescence. Collagen III was induced in the adipose stroma within 1 day, continued to increase through day 9 and was resolved by day 14. Immunoreactive tenascin was induced in the epithelium by day 1, was evident at the epithelial-stromal interface by day 5-9 and persisted as a condensed layer beneath the basement membrane through day 14. These findings parallel similar changes induced by gamma irradiation but demonstrate different onset and chronicity. In contrast, the integrity of epithelial basement membrane, which was unaffected by sparsely ionizing radiation, was disrupted by iron-particle irradiation. Laminin immunoreactivity was mildly irregular at 1 h postirradiation and showed discontinuities and thickening from days 1 to 9. Continuity was restored by day 14. Thus high-LET radiation, like sparsely ionizing radiation, induces rapid-remodeling of the stromal extracellular matrix but also appears to alter the integrity of the epithelial basement membrane, which is an important regulator of epithelial cell proliferation and differentiation.

NASA Discipline Radiation Health↗

NASA NTRS1994

Oscillating behavior of carbohydrate granule formation and dinitrogen fixation in the cyanobacterium Cyanothece sp. strain ATCC 51142

It has been shown that some aerobic, unicellular, diazotrophic cyanobacteria temporally separate photosynthetic O2 evolution and oxygen-sensitive N2 fixation. Cyanothece sp. ATCC strain 51142 is an aerobic, unicellular, diazotrophic cyanobacterium that fixes N2 during discrete periods of its cell cycle. When the bacteria are maintained under diurnal light-dark cycles, N2 fixation occurs in the dark. Similar cycling is observed in continuous light, implicating a circadian rhythm. Under N2-fixing conditions, large inclusion granules form between the thylakoid membranes. Maximum granulation, as observed by electron microscopy, occurs before the onset of N2 fixation, and the granules decrease in number during the period of N2 fixation. The granules can be purified from cell homogenates by differential centrifugation. Biochemical analyses of the granules indicate that these structures are primarily carbohydrate, with some protein. Further analyses of the carbohydrate have shown that it is a glucose polymer with some characteristics of glycogen. It is proposed that N2 fixation is driven by energy and reducing power stored in these inclusion granules. Cyanothece sp. strain ATCC 51142 represents an excellent experimental organism for the study of the protective mechanisms of nitrogenase, metabolic events in cyanobacteria under normal and stress conditions, the partitioning of resources between growth and storage, and biological rhythms.

Non-NASA Center↗

NASA NTRS2007

Dual-Beam Atom Laser Driven by Spinor Dynamics

An atom laser now undergoing development simultaneously generates two pulsed beams of correlated Rb-87 atoms. (An atom laser is a source of atoms in beams characterized by coherent matter waves, analogous to a conventional laser, which is a source of coherent light waves.) The pumping mechanism of this atom laser is based on spinor dynamics in a Bose-Einstein condensate. By virtue of the angular-momentum conserving collisions that generate the two beams, the number of atoms in one beam is correlated with the number of atoms in the other beam. Such correlations are intimately linked to entanglement and squeezing in atomic ensembles, and atom lasers like this one could be used in exploring related aspects of Bose-Einstein condensates, and as components of future sensors relying on atom interferometry. In this atom-laser apparatus, a Bose-Einstein condensate of about 2 x 10(exp 6) Rb-87 atoms at a temperature of about 120 micro-K is first formed through all-optical means in a relatively weak singlebeam running-wave dipole trap that has been formed by focusing of a CO2-laser beam. By a technique that is established in the art, the trap is loaded from an ultrahigh-vacuum magnetooptical trap that is, itself, loaded via a cold atomic beam from an upstream two-dimensional magneto-optical trap that resides in a rubidium-vapor cell that is differentially pumped from an adjoining vacuum chamber, wherein are performed scientific observations of the beams ultimately generated by the atom laser.

Thompson, Robert↗

NASA NTRS2010

Pathway Model of the Kinetics of the TGFbeta Antagonist Smad7 and Cross-Talk with the ATM and WNT Pathways

Signal transduction controls cellular and tissue responses to radiation. Transforming growth factor beta (TGFbeta) is an important regulator of cell growth and differentiation and tissue homeostasis, and is often dis-regulated in tumor formation. Mathematical models of signal transduction pathways can be used to elucidate how signal transduction varies with radiation quality, and dose and dose-rate. Furthermore, modeling of tissue specific responses can be considered through mechanistic based modeling. We developed a mathematical model of the negative feedback regulation by Smad7 in TGFbeta-Smad signaling and are exploring possible connections to the WNT/beta -catenin, and ATM/ATF2 signaling pathways. A pathway model of TGFbeta-Smad signaling that includes Smad7 kinetics based on data in the scientific literature is described. Kinetic terms included are TGFbeta/Smad transcriptional regulation of Smad7 through the Smad3-Smad4 complex, Smad7-Smurf1 translocation from nucleus to cytoplasm, and Smad7 negative feedback regulation of the TGFO receptor through direct binding to the TGFO receptor complex. The negative feedback controls operating in this pathway suggests non-linear responses in signal transduction, which are described mathematically. We then explored possibilities for cross-talk mediated by Smad7 between DNA damage responses mediated by ATM, and with the WNT pathway and consider the design of experiments to test model driven hypothesis. Numerical comparisons of the mathematical model to experiments and representative predictions are described.

Carra, Claudio↗

NASA NTRS2017

Effects of Ionizing Radiation on Murine Gene Expression in Skin and Bone

Long duration spaceflight causes a negative calcium balance and reduces bone density in astronauts. The potential for exposure to space radiation to contribute to lasting decrements in bone mass is not yet understood. Sustained changes to bone mass have a relatively long latency for development, however skin is a radiation sensitive organ and changes in skin gene expression may serve as an early radiation biomarker of exposures and may correlate with adverse effects on skeletal tissue. Previous studies have shown that FGF18 gene expression levels of hair follicles collected from astronauts on the ISS rose over time. In the hair follicle, FGF18 signaling mediates radioresistance in the telogen by arresting the cell cycle, and FGF18 has the potential to function as a radioprotector. In bone, FGF18 appears to regulate cell proliferation and differentiation positively during osteogenesis and negatively during chondrogenesis. Cellular defense responses to radiation are shared by a variety of organs, hence in this study, we examined whether radiation induced gene expression changes in skin may be predictive of the responses of skeletal tissue to radiation exposure. We have examined oxidative stress and growth arrest pathways in mouse skin and long bones by measuring gene expression levels via quantitative polymerase chain reaction (qPCR) after exposure to total body irradiation (TBI). To investigate the effects of irradiation on gene expression, we used skin and femora (cortical shaft) from the following treatment groups: control (normally loaded, sham-irradiated), and TBI (0.5 Gy Fe-56 600 MeV/n and 0.5 Gy H-1 150 MeV/n). Animals were euthanized one and 11 days post-IR. Statistical analysis was performed via a Student's ttest. In skin samples one day after IR, skin expression of FGF18 was significantly greater (3.8X) than sham-irradiated controls (3.8X), but did not differ 11 days post TBI. Expression levels of other radiation related genes (Nfe2l2, Trp53, Cdkn1a, FoxO3, Gadd45g, SOD1), was not different due to TBI at either time point. In bone (femora) TBI significantly increased (3.8X) expression of the pro-bone resorption cytokine, MCP-1, one day after TBI. FGF18 expression in skin and MCP- 1 expression in bone were found to be positively correlated (P less than 0.002, r=0.8779). Further, microcomputed tomography analysis of tibae from these animals showed reduced fractional cancellous bone volume (-21.7%) at 11 days post exposure. These results suggest that early radiation induced changes in FGF18 gene expression in skin may have value for predicting subsequent loss of cancellous bone mass.

space radiation↗

NASA NTRSDate not supplied

Optimizing Single Nuclei Sequencing of Brain Samples From Space Flown Mice Across Age and Strain

The NASA GeneLab Sample Processing Laboratory offers high-throughput sequencing services to NASA-funded space biology researchers. Space biology studies have specific challenges such as low sample numbers, introducing susceptibility to batch effects from sample handling. These issues are compounded by complex protocols such as single-nuclei isolation and sequencing, which has recently become an attractive methodology for assessing the cellular diversity within spaceflight samples. High quality single-nuclei sequencing requires reproducible protocols to dissociate tissue and generate clean suspension of intact single nuclei. Producing single-nuclei suspension from brain tissue is particularly challenging due to cell type heterogeneity and the myelin sheath that carries over into the nuclei suspension as debris. Current procedures tend to be time consuming and sometimes include steps that can alter gene expression and create cell-type bias. Commercially available nuclei isolation kits, such as the 10X Genomics nuclei isolation kit, offers a streamlined way to process samples for nuclei isolation, thereby minimizing batch effects and enabling reproducibility. In this study, we report on the performance of the 10X Genomics nuclei isolation kit and Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit to generate sequencing libraries from space-flown mouse brain samples. Single nuclei sequencing was performed on frozen mouse brain tissue from two spaceflight missions, Rodent Research-10 (RR-10) and RR Reference Mission-2 (RRRM-2). RR-10 mice were female B6129SF2/J, euthanized at 18-19 weeks whereas RRRM-2 mice were female C57BL/6NTac, euthanized at 20 or 37 weeks. Sequencing data was processed using standard GeneLab data processing pipelines. We report evaluation of the performance of the 10X Genomics nuclei isolation kit for spaceflight samples from mouse brain, and evaluation of reproducibility across different mouse strains and age groups. We also report preliminary scientific results including cell type inference, cell clustering, and differentially expressed genes and pathways between spaceflight and ground control samples.

RR-10↗

NASA NTRSDate not supplied

Gene Expression of Peripheral Blood Mononuclear Cells of Crew Members During Long-Duration Space Missions Indicate Dysregulation of Immunological and Cell Survival Mechanisms

Lymphocytes are naturally exposed to genotoxic stresses. DNA damage occurs during the entire lymphocyte’s life span and is induced mainly by reactive oxygen species (ROS), replication fork collapse, or telomere shortening during the immune response or intense cell proliferation phases. Strong evidence for the influence of immune function on DNA repair comes from studies of SCID disease. SCID mice not only have a deficient V(D)J recombination but are also unable to repair double strand breaks, leading to increased radiation sensitivity. The leukocytes’ transcriptome of 8 ISS crew members revels a dysregulated immune function and activation of cellular survival pathways in response to space environment. We have performed PCR analysis in peripheral mononuclear cells from the same crew members. A list of 62 genes were carefully selected addressing immunological and cell survival pathways. Differentially expressed genes indicated changes in chemokine receptor activity, chemokine binding, toll-like receptors, adhesion molecules and cellular response to DNA damage.

Maria Moreno-Villanueva↗

NASA NTRS2024

Gene Expression of Peripheral Blood Mononuclear Cells of Crew Members During Long-Duration Space Missions Indicate Dysregulation of Immunological and Cell Survival Mechanisms

María Moreno-Villanueva↗

NASA NTRS1996

Fibronectin regulates calvarial osteoblast differentiation

The secretion of fibronectin by differentiating osteoblasts and its accumulation at sites of osteogenesis suggest that fibronectin participates in bone formation. To test this directly, we determined whether fibronectin-cell interactions regulate progressive differentiation of cultured fetal rat calvarial osteoblasts. Spatial distributions of alpha 5 integrin subunit, fibronectin, osteopontin (bone sialoprotein I) and osteocalcin (bone Gla-protein) were similar in fetal rat calvaria and mineralized, bone-like nodules formed by cultured osteoblasts. Addition of anti-fibronectin antibodies to cultures at confluence reduced subsequent formation of nodules to less than 10% of control values, showing that fibronectin is required for normal nodule morphogenesis. Anti-fibronectin antibodies selectively inhibited steady-state expression of mRNA for genes associated with osteoblast differentiation; mRNA levels for alkaline phosphatase and osteocalcin were suppressed, whereas fibronectin, type I collagen and osteopontin were unaffected. To identify functionally relevant domains of fibronectin, we treated cells with soluble fibronectin fragments and peptides. Cell-binding fibronectin fragments (type III repeats 6-10) containing the Arg-Gly-Asp (RGD) sequence blocked both nodule initiation and maturation, whether or not they contained a functional synergy site. In contrast, addition of the RGD-containing peptide GRGDSPK alone did not inhibit nodule initiation, although it did block nodule maturation. Thus, in addition to the RGD sequence, other features of the large cell-binding fragments contribute to the full osteogenic effects of fibronectin. Nodule formation and osteoblast differentiation resumed after anti-fibronectin antibodies or GRGDSPK peptides were omitted from the media, showing that the inhibition was reversible and the treatments were not cytotoxic. Outside the central cell-binding domain, peptides from the IIICS region and antibodies to the N terminus did not inhibit nodule formation. We conclude that osteoblasts interact with the central cell-binding domain of endogenously produced fibronectin during early stages of differentiation, and that these interactions regulate both normal morphogenesis and gene expression.

NASA Program Space Physiology and Countermeasures↗

NASA NTRS1993

Gravity, chromosomes, and organized development in aseptically cultured plant cells

The objectives of the PCR experiment are: to test the hypothesis that microgravity will in fact affect the pattern and developmental progression of embryogenically competent plant cells from one well-defined, critical stage to another; to determine the effects of microgravity in growth and differentiation of embryogenic carrot cells grown in cell culture; to determine whether microgravity or the space environment fosters an instability of the differentiated state; and to determine whether mitosis and chromosome behavior are adversely affected by microgravity. The methods employed will consist of the following: special embryogenically competent carrot cell cultures will be grown in cell culture chambers provided by NASDA; four cell culture chambers will be used to grow cells in liquid medium; two dishes (plant cell culture dishes) will be used to grow cells on a semi-solid agar support; progression to later embryonic stages will be induced in space via crew intervention and by media manipulation in the case of liquid grown cell cultures; progression to later stages in case of semi-solid cultures will not need crew intervention; embryo stages will be fixed at a specific interval (day 6) in flight only in the case of liquid-grown cultures; and some living cells and somatic embryos will be returned for continued post-flight development and 'grown-out.' These will derive from the semi-solid grown cultures.

Krikorian, Abraham D.↗

DOE OSTI2025

Machine learning models for segmentation and classification of cyanobacterial cells

Abstract Timelapse microscopy has recently been employed to study the metabolism and physiology of cyanobacteria at the single-cell level. However, the identification of individual cells in brightfield images remains a significant challenge. Traditional intensity-based segmentation algorithms perform poorly when identifying individual cells in dense colonies due to a lack of contrast between neighboring cells. Here, we describe a newly developed software package called Cypose which uses machine learning (ML) models to solve two specific tasks: segmentation of individual cyanobacterial cells, and classification of cellular phenotypes. The segmentation models are based on the Cellpose framework, while classification is performed using a convolutional neural network named Cyclass. To our knowledge, these are the first developed ML-based models for cyanobacteria segmentation and classification. When compared to other methods, our segmentation models showed improved performance and were able to segment cells with varied morphological phenotypes, as well as differentiate between live and lysed cells. We also found that our models were robust to imaging artifacts, such as dust and cell debris. Additionally, the classification model was able to identify different cellular phenotypes using only images as input. Together, these models improve cell segmentation accuracy and enable high-throughput analysis of dense cyanobacterial colonies and filamentous cyanobacteria.

Huffine, Clair A.↗

NASA NTRS1981

On the magnetic viscosity in Keplerian accretion disks

The paper develops a model for the anomalous viscosity in accretion disks based on the hypothesis that the hydrodynamic turbulence within the disk takes the form of spatially localized magnetic flux cells. The local shear flow due to Keplerian differential rotation distorts the flux cell topology, converting shear flow energy into magnetic energy. In the radial diffusion approximation, the kinematic viscosity is estimated from the radial displacement and is shown to maximize at flux cell scale lengths for which the shear flow stopping and reconnection times are equal.

Coroniti, F. V.↗

NASA NTRS1983

Static Feed Water Electrolysis Subsystem Testing and Component Development

A program was carried out to develop and test advanced electrochemical cells/modules and critical electromechanical components for a static feed (alkaline electrolyte) water electrolysis oxygen generation subsystem. The accomplishments were refurbishment of a previously developed subsystem and successful demonstration for a total of 2980 hours of normal operation; achievement of sustained one-person level oxygen generation performance with state-of-the-art cell voltages averaging 1.61 V at 191 ASF for an operating temperature of 128F (equivalent to 1.51V when normalized to 180F); endurance testing and demonstration of reliable performance of the three-fluid pressure controller for 8650 hours; design and development of a fluid control assembly for this subsystem and demonstration of its performance; development and demonstration at the single cell and module levels of a unitized core composite cell that provides expanded differential pressure tolerance capability; fabrication and evaluation of a feed water electrolyte elimination five-cell module; and successful demonstration of an electrolysis module pressurization technique that can be used in place of nitrogen gas during the standby mode of operation to maintain system pressure and differential pressures.

Koszenski, E. P.↗

NASA NTRS1983

Short-term and long-term clinostat and vibration-induced biochemical changes in dwarf marigold stems

Stems of 21-day dwarf marigold plants cultivated on the clinostat were compared with plants cultivated on vertical axis rotators ('vibrational controls') and stationary controls for long-term changes in cell wall composition. Stems of 21-day plants grown under stationary conditions and subsequently exposed to the clinostat for 24 hours were also analyzed. Among the long-term markers, calcium, lignin, and protein-bound hemicellulose (possibly cell wall glycoprotein) clearly differentiated the effects of vibration from those of the clinostat. Short-term differential responses included rate of ethylene production, nastic movement and peroxidase activity of the cell wall, but not of the protoplast.

Siegel, S. M.↗

NASA NTRS1998

Three-dimensional culture of rat calvarial osteoblasts in porous biodegradable polymers

Neonatal rat calvarial osteoblasts were cultured in 90% porous, 75:25 poly(DL-lactic-co-glycolic acid) (PLGA) foam scaffolds for up to 56 days to examine the effects of the cell seeding density, scaffold pore size, and foam thickness on the proliferation and function of the cells in this three-dimensional environment. Osteoblasts were seeded at either 11.1 x 10(5) or 22.1 x 10(5) cells per cm2 onto PLGA scaffolds having pore sizes in the range of 150-300 or 500-710 microm with a thickness of either 1.9 or 3.2 mm. After 1 day in culture, 75.6 and 68.6% of the seeded cells attached and proliferated on the 1.9 mm thick scaffolds of 150-300 microm pore size for the low and high seeding densities, respectively. The number of osteoblasts continued to increase throughout the study and eventually leveled off near 56 days, as indicated by a quantitative DNA assay. Osteoblast/foam constructs with a low cell seeding density achieved comparable DNA content and alkaline phosphatase (ALPase) activity after 14 days, and mineralization results after 56 days to those with a high cell seeding density. A maximum penetration depth of osseous tissue of 220+/-40 microm was reached after 56 days in the osteoblast/foam constructs of 150-300 microm pore size initially seeded with a high cell density. For constructs of 500-710 microm pore size, the penetration depth was 190+/-40 microm under the same conditions. Scaffold pore size and thickness did not significantly affect the proliferation or function of osteoblasts as demonstrated by DNA content, ALPase activity, and mineralized tissue formation. These data show that comparable bone-like tissues can be engineered in vitro over a 56 day period using different rat calvarial osteoblast seeding densities onto biodegradable polymer scaffolds with pore sizes in the range of 150-710 microm. When compared with the results of a previous study where similar polymer scaffolds were seeded and cultured with marrow stromal cells, this study demonstrates that PLGA foams are suitable substrates for osteoblast growth and differentiated function independent of cell source.

Non-NASA Center↗

NASA NTRS1999

Differential expression of human lysyl hydroxylase genes, lysine hydroxylation, and cross-linking of type I collagen during osteoblastic differentiation in vitro

The pattern of lysyl hydroxylation in the nontriple helical domains of collagen is critical in determining the cross-linking pathways that are tissue specific. We hypothesized that the tissue specificity of type I collagen cross-linking is, in part, due to the differential expression of lysyl hydroxylase genes (Procollagen-lysine,2-oxyglutarate,5-dioxygenase 1, 2, and 3 [PLOD1, PLOD2, and PLOD3]). In this study, we have examined the expression patterns of these three genes during the course of in vitro differentiation of human osteoprogenitor cells (bone marrow stromal cells [BMSCs]) and normal skin fibroblasts (NSFs). In addition, using the medium and cell layer/matrix fractions in these cultures, lysine hydroxylation of type I collagen alpha chains and collagen cross-linking chemistries have been characterized. High levels of PLOD1 and PLOD3 genes were expressed in both BMSCs and NSFs, and the expression levels did not change in the course of differentiation. In contrast to the PLOD1 and PLOD3 genes, both cell types showed low PLOD2 gene expression in undifferentiated and early differentiated conditions. However, fully differentiated BMSCs, but not NSFs, exhibited a significantly elevated level (6-fold increase) of PLOD2 mRNA. This increase coincided with the onset of matrix mineralization and with the increase in lysyl hydroxylation in the nontriple helical domains of alpha chains of type I collagen molecule. Furthermore, the collagen cross-links that are derived from the nontriple helical hydroxylysine-aldehyde were found only in fully differentiated BMSC cultures. The data suggests that PLOD2 expression is associated with lysine hydroxylation in the nontriple helical domains of collagen and, thus, could be partially responsible for the tissue-specific collagen cross-linking pattern.

NASA Discipline Musculoskeletal↗

NASA NTRS1998

Formation and differentiation of three-dimensional rat marrow stromal cell culture on microcarriers in a rotating-wall vessel

Using a high aspect ratio vessel (HARV), this study investigated the formation of 3-D rat marrow stromal cell culture on microcarriers and the expression of bone-related biochemical markers under conditions of simulated microgravity. In addition, it calculated the shear stresses imparted on the surface of microcarriers of different densities by the medium fluid in an HARV. Secondary rat marrow stromal cells were cultured on two types of microcarriers, Cytodex-3 beads and modified bioactive glass particles. Examination of cellular morphology by scanning electron microscopy revealed the presence of three-dimensional multicellular aggregates consisting of multiple cell-covered Cytodex-3 microcarriers bridged together. Mineralization was observed in the aggregates. Spherical cell-bead aggregates were observed in an HARV, while cell-bead assemblies were mostly loosely packed in a chain-like or branched structure in a cell bag. The expressions of alkaline phosphatase activity, collagen type I, and osteopontin were shown via the use of histochemical staining, immunolabeling, and confocal scanning electron microscopy. Using a numerical approach, it was found that at a given rotational speed and for a given culture medium, a larger density difference between the microcarrier and the culture medium (e.g., a modified bioactive glass particle) imparted a higher maximum shear stress on the microcarrier.

Dextrans↗