Agarose Mold Research Articles

Three-Dimensional Model Analysis Revealed Differential Cytotoxic Effects of the NK-92 Cell Line and Primary NK Cells on Breast and Ovarian Carcinoma Cell Lines Mediated by Variations in Receptor-Ligand Interactions and Soluble Factor Profiles.

Background/objectives: The functional activity of a certain tumor determines the effectiveness of primary NK cells and NK-92 cell line-based cancer therapy; their therapeutic effectiveness against different tumors can vary. This work provides a direct simultaneous comparison of the cytotoxic effects of in vitro-activated peripheral NK (pNK) cells and NK-92 cells in spheroid models of BT-474, MCF7 and SKOV-3 carcinomas and uncovers the reasons for the differential effectiveness of NK cells against tumors. Methods: Tumor spheroids of similar size and shape, obtained from agarose molds, were incubated with NK-92 or pNK cells for 24 h. Tumor cell death was detected using flow cytometry or confocal microscopy. Cytokine production, granzyme B levels and NK cell degranulation analyses were performed, along with pNK and target-cell phenotypic characterization. Results: While NK-92 and pNK cells lysed BT-474 spheroids with comparably low efficiency, pNK cells were more capable of eliminating MCF7 and SKOV-3 spheroids than NK-92 cells were. The results of the functional and phenotypic analyses strongly support the participation of the NKG2D-NKG2DL pathway in pNK cell activation induced by the most sensitive cytotoxic attack on SKOV-3 spheroids, whereas the CX3CR1-CX3CL1 axis appears to be involved in the pNK reaction against MCF-7 spheroids. Conclusions: We provide a new approach for the preliminary identification of the most promising NK cell receptors that can alter the effectiveness of cancer therapy depending on the specific tumor type. Using this approach, NK-92 cells or pNK subsets can be selected for further accumulation and/or genetic modification to improve specificity and reactivity.

Novel 3D human trophoblast culture to explore T. cruzi infection in the placenta.

Human trophoblastic cell lines, such as BeWo, are commonly used in 2D models to study placental Trypanosoma cruzi infections. However, these models do not accurately represent natural infections. Three-dimensional (3D) microtissue cultures offer a more physiologically relevant in vitro model, mimicking tissue microarchitecture and providing an environment closer to natural infections. These 3D cultures exhibit functions such as cell proliferation, differentiation, morphogenesis, and gene expression that resemble in vivo conditions. We developed a 3D culture model using the human trophoblastic cell line BeWo and nonadherent agarose molds from the MicroTissues® 3D Petri Dish® system. Both small (12-256) and large (12-81) models were tested with varying initial cell numbers. We measured the diameter of the 3D cultures and evaluated cell viability using Trypan Blue dye. Trophoblast functionality was assessed by measuring β-hCG production via ELISA. Cell fusion was evaluated using confocal microscopy, with Phalloidin or ZO-1 marking cell edges and DAPI staining nuclei. T. cruzi infection was assessed by microscopy and quantitative PCR, targeting the EF1-α gene for T. cruzi and GAPDH for BeWo cells, using three parasite strains: VD (isolated from a congenital Chagas disease infant and classified as Tc VI), and K98 and Pan4 (unrelated to congenital infection and classified as Tc I). Seeding 1000 BeWo cells per microwell in the large model resulted in comparable cellular viability to 2D cultures, with a theoretical diameter of 408.68 ± 12.65 μm observed at 5 days. Functionality, assessed through β-hCG production, exceeded levels in 2D cultures at both 3 and 5 days. T. cruzi infection was confirmed by qPCR and microscopy, showing parasite presence inside the cells for all three tested strains. The distribution and progression of the infection varied with each strain. This innovative 3D model offers a simple yet effective approach for generating viable and functional cultures susceptible to T. cruzi infection, presenting significant potential for studying the placental microenvironment.

Biofabrication of Heterogeneous, Multi‐Layered, and Human‐Scale Tissue Transplants Using Eluting Mold Casting

AbstractThe creation of multi‐tissue auricular transplants for the treatment of microtia is a challenge due to the complex and layered structure of this anatomical tissue. A novel casting technique for the 3D biofabrication of heterogeneous, multi‐layered, and human‐scale tissue transplants using eluting agarose molds is presented. The molds are generated by casting agarose into custom 3D‐printed containers, termed metamolds, optimized to facilitate the hydrogel casting process based on geometric and topological constraints. Casting yields high resolution (50 µm) and allows for subsequent casting of further hydrogel layers on the transplant. Multi‐layered auricular constructs are fabricated on a cartilage core consisting of a hyaluronic acid‐alginate double network and an adjacent gelatin‐based dermal layer. Bonding between adjacent layers is achieved by orthogonal physical and enzymatic crosslinking of residual functional groups between each layer. Material composition and culture duration are optimized for each layer allowing for maturation into cartilaginous and pre‐vascularized dermal tissues. To demonstrate the scalability of this technique for the biofabrication of human‐sized transplants, bi‐layered human‐sized ears are cast. Overall, this novel casting technique offers a promising approach for the fabrication of complex tissue grafts, overcoming the limitations of other traditional biofabrication methods.

HIGH-RESOLUTION 3D-PRINTED MICROSCAFFOLDS FOR BOTTOM-UP CARTILAGE TISSUE ENGINEERING

In the field of tissue engineering (TE), mainly two approaches have been widely studied and utilised throughout the last two decades. Ovsianikov et al. proposed a third strategy for tissue engineering to combine the advantages of the scaffold-based and scaffold-free approach [1].We utilise the third strategy for TE by fabrication of cell spheroids that are reinforced by microscaffolds, called tissue units (TUs). Aim of the presented study is to differentiate TUs towards a chondrogenic phenotype to show the self-assembly of a millimetre sized cartilage-like tissue in a bottom-up TE approach in vitro.Two-Photon polymerization (2PP) was utilised to fabricate highly porous microscaffolds with a diameter of 300 µm. The biocompatible and biodegradable, resin Degrad INX (supplied from Xpect INX, Ghent, Belgium) was used for 3D-printing. Each microscaffold was seeded with 4000 human adipose derived stem cells (hASCs) in low-adhesive 96-well plates to allow spheroid formation. TUs were differentiated towards the chondrogenic lineage by application of chondrogenic media, subsequently merged in a cylindrical agarose mold, to fuse into a connected tissue with a diameter of ~1.8 mm and a height of 8 mm.The characterization of TUs differentiated towards the chondrogenic phenotype included gene expression and protein analysis. Furthermore, immunohistochemically staining for Collagen II and Alcian blue staining were performed to investigate the matrix deposition and fusion of the self-assembled tissue.Our results suggest that the utilised method could be a promising approach for a variety of tissue engineering approaches, due to the good applicability to a defect side combined with the self-assembly properties of the TUs. Furthermore, the differentiation potential of hASCs is not limited to chondrogenic lineages only, which could pave the way to further TE applications in the future.Acknowledgements:This research work was financially supported by the European Research Council (Consolidator Grant 772464 A.O.)

FC076: Development of a High-Throughput in Vivo Drug Screening Assay Using a FSGS-Like Zebrafish Model

Abstract BACKGROUND AND AIMS Focal segmental sclerosis (FSGS) is a severe histopathological condition which often results in end-stage renal disease. Loss of podocytes and severe changes of podocyte foot process morphology accompanied by proteinuria are the hallmarks of FSGS. Since no therapeutic agents are available until now, further efforts must be made to identify new drugs to treat this disease. The zebrafish larva is a widely used kidney research model as the morphology and function of zebrafish and mammalian glomeruli are very similar. Since our group has already established a FSGS-like zebrafish injury model based on the nitroreductase-metronidazole cell ablation system, we have developed a high-throughput based in vivo drug screening assay. METHOD We used a zebrafish screening strain that expresses mCherry and the bacterial enzyme nitroreductase in podocytes. Additionally, a circulating vitamin-D-binding eGFP fusion protein with a size of 78 kDa is expressed and serves as a read-out for proteinuria. Incubation with 80 µM metronidazole for 24 h at 4 days post-fertilization (dpf) induces an FSGS-like phenotype in these larvae. A library of 138 epigenetic drugs were screened and the results are presented herein. Groups of 12 larvae received injury induction as well as drug treatment and were transferred individually into 96-well plates prepared with custom agarose molds. Larvae were oriented laterally and imaged with a high-content screening microscope (Acquifer Imaging Machine). The eGFP signal in the caudal vasculature as well as the glomerular mCherry signal served as readout systems. At 6 dpf, the same larvae and regions were imaged again. Custom written FIJI codes automatically segmented and measured the fluorescence intensities of both readouts and timepoints. For each larva, intensity ratios were determined and the ratios of each treatment group served as the input for statistical analysis. RESULTS Treatment for 24 hours with 80 µM metronidazole resulted in a measurable decline of the eGFP signal in the vasculature and of the podocyte mCherry signal compared to the vehicle (DMSO) group. Simultaneous treatment of metronidazole with the 138 drugs in the epigenetics library resulted in beneficial outcomes in varying degrees (9 drugs were positive, 36 compounds were lethal). CONCLUSION This new in vivo semi-automated high-throughput compatible drug screening assay allows rapid identification of FSGS protective drugs. Here we screened 138 epigenetic drugs, which led to the identification of 9 potential drugs which have had a beneficial effect on podocytes and/or the glomerular filtration barrier.

Quantifying Cell-Derived Changes in Collagen Synthesis, Alignment, and Mechanics in a 3D Connective Tissue Model.

Dysregulation of extracellular matrix (ECM) synthesis, organization, and mechanics are hallmark features of diseases like fibrosis and cancer. However, most in vitro models fail to recapitulate the three‐dimensional (3D) multi‐scale hierarchical architecture of collagen‐rich tissues and as a result, are unable to mirror native or disease phenotypes. Herein, using primary human fibroblasts seeded into custom fabricated 3D non‐adhesive agarose molds, a novel strategy is proposed to direct the morphogenesis of engineered 3D ring‐shaped tissue constructs with tensile and histological properties that recapitulate key features of fibrous connective tissue. To characterize the shift from monodispersed cells to a highly‐aligned, collagen‐rich matrix, a multi‐modal approach integrating histology, multiphoton second‐harmonic generation, and electron microscopy is employed. Structural changes in collagen synthesis and alignment are then mapped to functional differences in tissue mechanics and total collagen content. Due to the absence of an exogenously added scaffolding material, this model enables the direct quantification of cell‐derived changes in 3D matrix synthesis, alignment, and mechanics in response to the addition or removal of relevant biomolecular perturbations. To illustrate this, the effects of nutrient composition, fetal bovine serum, rho‐kinase inhibitor, and pro‐ and anti‐fibrotic compounds on ECM synthesis, 3D collagen architecture, and mechanophenotype are quantified.

Tissue Engineering of Canine Cartilage from Surgically Debrided Osteochondritis Dissecans Fragments

This study in dogs explored the feasibility of using cartilage fragments removed and discarded during routine palliative surgery for osteochondritis dissecans (OCD) as a source of primary chondrocytes for scaffold-free cartilage tissue-engineering. Primary chondrocytes were obtained from three OCD donors and one age-matched healthy articular cartilage (HAC) donor. After monolayer expansion of primary cells, a three-dimensional spherical suspension culture was implemented. Following this stage, cells were seeded at a high density into custom-made agarose molds that allowed for size and shape-specific constructs to be generated via a method of cellular self-assembling in a scaffold-free environment. Fifty-eight neocartilage constructs were tissue-engineered using this methodology. Neocartilage constructs and native cartilage from shoulder joint were subjected to histological, mechanical, and biochemical testing. OCD and HAC chondrocytes-sourced constructs had uniformly flat morphology and histology consistent with cartilage tissue. Constructs sourced from OCD chondrocytes were 1.5-times (32%) stiffer in compression and 1.3 times (23%) stronger in tension than constructs sourced from HAC chondrocytes and only 8.7-times (81%) less stiff in tension than native tissue. Constructs from both cell sources consistently had lower collagen content than native tissue (22.9%/dry weight [DW] for OCD and 4.1%/DW for HAC vs. 51.1%/DW native tissue). To improve the collagen content and mechanical properties of neocartilage, biological and mechanical stimuli, and thyroid hormone (tri-iodothyronine) were applied to the chondrocytes during the self-assembling stage in two separate studies. A 2.6-fold (62%) increase in compressive stiffness was detected with supplementation of biological stimuli alone and 5-fold (81%) increase with combined biological and mechanical stimuli at 20% strain. Application of thyroid hormone improved collagen content (1.7-times, 33%), tensile strength (1.8-times, 43%), and stiffness (1.3-times, 21%) of constructs, relative to untreated controls. Collectively, these data suggest that OCD chondrocytes can serve as a reliable cell source for cartilage tissue-engineering and that canine chondrocytes respond favorably to biological and mechanical stimuli that have been shown effective in chondrocytes from other animal species, including humans.

A Method for the Evaluation of Early Osseointegration of Implant Materials Ex Vivo: Human Bone Organ Model.

In the present work, an ex vivo organ model using human bone (explant) was developed for the evaluation of the initial osseointegration behavior of implant materials. The model was tested with additive manufactured Ti6Al4V test substrates with different 3D geometries. Explants were obtained from patients who underwent total knee replacement surgery. The tibial plateaus were used within 24 h after surgery to harvest bone cylinders (BC) from the anterior side using hollow burrs. The BCs were brought into contact with the test substrate and inserted into an agarose mold, then covered with cell culture media and subjected to the external load of 500 g. Incubation was performed for 28 days. After 28d the test substrate was removed for further analysis. Cells grown out BC onto substrate were immunostained with DAPI and with an antibody against Collagen-I and alkaline phosphatase (ALP) for visualization and cell counting. We show that cells stayed alive for up to 28d in our organ model. The geometry of test substrates influences the number of cells grown onto substrate from BCs. The model presented here can be used for testing implant materials as an alternative for in vitro tests and animal models.

PHOTOBIOMODULATORY EFFECT OF LOW-INTENSITY LASER RADIATION ON MULTICELLULAR SPHEROIDS

Photobiomodulatory effects of low-intensity laser radiation (LILR) in cells cultured in standard, two-dimensional conditions are well established. Conversely, the characteristics of this effect in three-dimensional (3D) cultures, which are currently recommended due to the greater similarity with cellular behavior in vivo, have not yet been widely investigated. The objective of this work was to analyze the biomodulator effect of LILR, on the wavelength (λ) of 685 nm, on the constitution process and on the viability of cells cultured as multicellular spheroid (MSs). For this, agarose molds containing microwells were seeded (2x105 cell/ml) with osteogenic precursor cells (OPCs - MC3T3-E1) and kept under ideal culture conditions. The molds were irradiated for five consecutive days with doses of 0.5, 1.0 and 1.5 J/cm², the first irradiation being performed immediately after sowing. The process of constitution of MSs was analyzed and the cultures were submitted to the cell viability test. The results demonstrated that the LILR at λ 685 nm exerted a dose-dependent biomodulatory effect on cell metabolism and on the process of constituting the MSs of OPCs. These results demonstrate the potential of photobiomodulation to contribute to the process of constituting MSs, which can be explored in the strategies of multicellular spheroids therapy used in regenerative medicine and bioprinting.

Efficient manufacturing of tissue engineered cartilage in vitro by a multiplexed 3D cultured method.

Cell culture has become an indispensable tool to uncover fundamental biophysical and biomolecular mechanisms of cells assembling into tissues. An important advancement in cell culture techniques was the introduction of three-dimensional (3D) culture systems. In this study, the mutual fusion of chondrocyte pellets was promoted in order to produce large-sized tissue-engineered cartilage by a multiplexed 3D hanging drop culture and agarose mold method to optimize the means of cultivation. Cell proliferation, aggregation, cell morphology maintenance as well as cartilage related gene expression and matrix secretion in vitro and subcutaneous implantation models were evaluated. These results indicated that the multiplexed 3D hanging drop culture involving the fusion of small pellets into a large structure enabled the efficient production of 3D tissue engineered cartilage that was closer to physiological cartilage tissue in comparison to that of the agarose mold method.

Relationship Between Ex Vivo Tissue Diffusion Tensor Indexes and Material Properties in Posterior Tibialis Tendon

Category:Ankle, tendon imagingIntroduction/Purpose:Posterior tibialis tendon is of clinical importance in the development and progression of acquired flatfoot and other midfoot deformity. The ability to quantitatively evaluate tendon tissue non-invasively would enable assessment of tendon health status and tracking of recovery from injury. Magnetic resonance diffusion tensor imaging (DTI) has been used to examine tendon tissue organization in healing tendon tissue. However, the relationship of DTI-based measures to tendon mechanical function has not been established. The purpose of this pilot study was to quantitatively evaluate posterior tibialis tendon using DTI and determine the relationship of these parameters to tendon function assessed via ex vivo mechanical testing.Methods:Posterior tibialis tendons from individuals undergoing amputation were positioned vertically in an agarose mold filled with saline for imaging. High resolution diffusion imaging parameters were optimized for tendon on a 3 T MRI to acquire 13 6-mm transverse slices covering the length: 1mm2 isotropic resolution, 2 signal averaging, repetition/echo times of 5000/58 ms, diffusion strength of 500s/mm2 with 30 gradient directions, scan time 5 min. Diffusion images had sufficient quality and were corrected for motion and image distortion. DTI parametric maps including fractional anisotropy (FA), mean, axial, and radial diffusivities (MD, AD, and RD; mm2/s) were calculated along with fiber tracking indexes of fiber length (mm) and density. After imaging, specimens were preloaded to 10 Newtons, preconditioned 10 cycles at 6% strain, subjected to stress-relaxation at 6% strain (10 minutes), then loaded to a maximum of 10% strain. Relationships between DTI indexes and mechanical properties (stiffness and hysteresis) were evaluated using Spearman correlation.Results:Six individuals (4 male, mean(SD) age: 56(5)years, body mass index: 30(6) kg/m2) were included. Reason for amputation was diabetes-related complications in 5 participants and failed orthopaedic surgery in 1 participant. In DTI (Figure 1A), tendons had a tract length of 11.5(11.3)mm and tract density of 23.9(2.4) per ROI. FA, MD, AD, and RD quantify how freely a water molecule is able to move within the tissue, and the directionality of that movement. Tendons had an FA of 0.26(0.25), MD of 1.25(1.28), AD of 1.54(1.57), and RD of 1.11(1.14). Tract length was positively related to linear stiffness (rho=0.829, p=0.04) and hysteresis at 10% strain (rho=0.886, p=0.019) (Figure 1B-D). AD was positively related to hysteresis at 10% strain (rho=0.812, p=0.05).Conclusion:This is the first study to describe posterior tibialis tendon appearance on DTI. Tract length and AD are both related to tendon mechanics. Tract length is based on quantity and directionality of water displacement and may indicate degree of collagen organization given its relationship to stiffness. Tract length and AD related positively to hysteresis, which will require additional research to identify the mechanisms behind this relationship. This study is limited by sample size and specimens that likely do not represent healthy tissue. Regardless, these findings support continued investigation into in vivo imaging of tendon with DTI for quantitative tendon assessment.

Rapid Electroformation of Biopolymer Gels in Prescribed Shapes and Patterns: A Simpler Alternative to 3-D Printing.

We demonstrate the use of electric fields to rapidly form gels of the biopolymer alginate (Alg) in specific three-dimensional (3-D) shapes and patterns. In our approach, we start with a gel of the biopolymer agarose, which is thermoresponsive and hence can be molded into a specific shape. The agarose mold is then loaded with Ca2+ cations and placed in a beaker containing an Alg solution. The inner surface of the beaker is surrounded by aluminum foil (cathode), and a copper wire (anode) is stuck in the agarose mold. These are connected to a direct current (DC) power source, and when a potential of ∼10 V is applied, an Alg gel is formed in a shape that replicates the mold. Gelation occurs because the Ca2+ ions electrophoretically migrate away from the mold, whereupon they cross-link the Alg chains adjacent to the mold. At low Ca2+ (0.01 wt %), the Alg gel layer grows outward from the mold surface at a steady rate of about 0.8 mm/min, and the gel stops growing when the field is switched off. After a gel of desired thickness is formed, the agarose mold can be melted away to leave behind an Alg gel in a precise shape. Alg gels formed in this manner are transparent and robust. This process is particularly convenient to form Alg gels in the form of hollow tubes, including tubes with multiple concentric layers, each with a different payload. The technique is safe for encapsulation of biological species within a given Alg layer. We also create Alg gels in specific patterns by directing gel growth around selected regions. Overall, our technique enables lab-scale manufacturing of alginate gels in 3-D without the need for an expensive 3-D printer.

Contraction dynamics of\xa0dental pulp cell rod\xa0microtissues

ObjectivesThe factors that contribute to the morphological changes of dental pulp cell–derived microtissues are unknown. Here, we investigated the contraction dynamics of rod-shaped microtissues derived from dental pulp cells and examined the underlying cell signaling pathways.MethodsHuman dental pulp cells were seeded into agarose molds to assemble into rod-shaped microtissues. Resazurin- and tetrazolium-based cytotoxicity assays, Live/Dead staining, and hematoxylin and eosin staining for histological evaluation of rods were performed. Rod contraction was evaluated and measured for a period of 10 days. The role of TGF-β, phosphoinositide 3-kinase (PI3K)/AKT, and mitogen-activated protein kinase (MAPK) signaling pathway was analyzed.ResultsDental pulp cells readily assembled into rods, maintaining the geometric shape for 48 h. Following this period, they condensed to form stable spheroidal structures that remained vital for 10 days from seeding. Inhibition of phosphoinositide 3-kinase signaling pathway by LY294002 significantly prolonged the diminution in the length of rods formed by dental pulp cells. TGF-β and pharmacological inhibition of TGF-β signaling did not show pronounced effects.ConclusionOverall, dental pulp cells readily formed rod-shaped patterns of microtissues which, over a period of time, condensed into more stable spheroidal structures. Hence, technologies like bioprinting, using direct fabrication of microtissues need to consider the contraction dynamics.Clinical relevanceThe field of regenerative endodontology will benefit from our findings as it can be applied as a novel platform to test the impact of pharmacological agents, biomaterials, and regenerative approaches including bioprinting.

Biostimulating Effect of 685 nm Low‐Intensity Laser Radiation on Cell Viability in Multicellular Spheroids Culture

Recent research evidences substantial morphological and physiological differences between cells maintained in vitro in two‐dimensional (2D) and three‐dimensional (3D) culture conditions. The current consensus is that 3D culture is better because it precisely mimics the cellular microenvironment in vivo. The biomodulatory effect of low‐intensity laser radiation (LILR) on cells in 2D cultures is already well established. On the other hand, the effect of this radiation on cells in 3D cultures has not yet been widely investigated. The aim of this work was to analyze the biomodulatory effect of LILR, at the wavelength (ƛ) of 685 nm, on the viability of osteogenic precursor cells (OPC) cultured as multicellular spheroids (MS). Agarose molds containning wells (300 μm diameter) were used for MS formation using the non‐adherent surface culture concept. A cell count (2×105) of the OPC lineage (MC3T3) was seeded in each mold and maintained under ideal growth conditions (Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum/37°C/95% humidified atmospheric air/5% CO2). The molds were irradiated for 5 consecutive days at doses of 0.5, 1.0 or 1.5 J/cm2 (triplicates), and the first irradiation was performed immediately after seeding. After this period, the cultures were submitted to cell viability assay and the results were expressed as a percentage of non‐irradiated control cultures (ANOVA ONE‐WAY, p≤0.05). The results showed that LILR, at ƛ 685 nm, provided a 79% increase in cell viability at the dose of 1J/cm2. The remaining doses exhibited similar viability rates that the control. These results confirm the dose‐dependent biostimulatory effect of LILR at ƛ 685 nm on MS culture and contribute to the understanding of the real influence of this radiation on cells under ideal conditions for in vitro simulation studies.Support or Funding InformationCoordenação de Aperfeiçoamento de Pessoal de Nível Superior ‐ CAPES ‐ BrazilThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Contraction Dynamics of Rod Microtissues of Gingiva-Derived and Periodontal Ligament-Derived Cells.

Tissue engineering strategies using microtissues as “building blocks” have high potential in regenerative medicine. Cognition of contraction dynamics involved in the in vitro self-assembly of these microtissues can be conceived as the bedrock of an effective periodontal tissue regenerative therapy. Our study was directed at evaluating the shrinkage in the rod-shaped structure of a directed self-assembly of human gingiva-derived cells (GC) and periodontal ligament-derived cells (PDLC) and developing insights into the potential mechanisms responsible for the shrinkage. GC and PDLC were seeded in non-adherent agarose molds to form rod microtissues. Cells used for the experiments were characterized using fluorescence-activated cell sorting (FACS). To assess the viability, resazurin-based cytotoxicity assays, trypan blue dye exclusion assay, MTT and live/dead staining, and histological evaluation of rods based on hematoxylin and eosin staining were performed. Rod contraction was evaluated and measured at 0, 2, 6, and 24 h and compared to L-929 cells. The role of transforming growth factor (TGF)-β signaling, phosphoinositide 3-kinase (PI3K)/AKT, and mitogen activated protein kinase (MAPK) signaling was analyzed. Our results show that the rod microtissues were vital after 24 h. A reduction in the length of rods was seen in the 24 h period. While the recombinant TGF-β slightly reduced contraction, inhibition of TGF-β signaling did not interfere with the contraction of the rods. Interestingly, inhibition of phosphoinositide 3-kinase by LY294002 significantly delayed contraction in GC and PDLC rods. Overall, GC and PDLC have the ability to form rod microtissues which contract over time. Thus, approaches for application of these structures as “building blocks” for periodontal tissue regeneration should consider that rods have the capacity to contract substantially. Further investigation will be needed to unravel the mechanisms behind the dynamics of contraction.

Adherent agarose mold cultures: An in vitro platform for multi-factorial assessment of passaged chondrocyte redifferentiation.

Generating the best possible bioengineered cartilage from passaged chondrocytes requires culture condition optimization. In this study, the use of adherent agarose mold (adAM) cultures to support redifferentiation of passaged twice (P2) chondrocytes and serve as a scalable platform to assess the effect of growth factor combinations on proteoglycan accumulation by cells was examined. By 2 days in adAM culture, bovine P2 cells were partially redifferentiated as demonstrated by regression of actin-based dedifferentiation signalling and fibroblast matrix and contractile gene expression. By day 10, aggrecan and type II collagen gene expression were significantly increased in adAM cultured cells. At day 20, a continuous layer of cartilage tissue was observed. There was no evidence of tissue contraction by P2 cells in adAM cultures. The matrix properties of the resultant tissue as well as proteoglycan 4 (PRG4) secreted by the cells were dependent on the initial cell seeding density. AdAM cultures were scalable and culture within small 3 mm diameter adAM allowed for multi-factorial assessment of growth factors on proteoglycan accumulation by human P2 chondrocytes. Although there was a patient specific response in proteoglycan accumulation to the various cocktail combinations, the cocktail consisting of 2 ng/ml TGFβ1, 10 ng/ml FGF2, and 250 ng/ml FGF18 resulted in a consistent increase in alcian blue tissue staining. Additional studies will be required to identify the optimal conditions to bioengineer articular cartilage tissue for clinical use. However, the results to date suggest that adAM cultures may be suitable to use for high throughput assessment. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2392-2405, 2018.

Down-regulation of PAX2 promotes in vitro differentiation of podocytes from human CD34+ cells.

Podocytes are major kidney cells that help in glomerular filtration and any damage or loss is a major event in the progression of kidney diseases. Understanding podocytes development will help in designing therapeutic strategies against these renal diseases. Therefore, in vitro generation of podocytes from adult hematopoietic CD34+ stem cells is explored in the present study. Apheretically, isolated human HSCs from peripheral blood showed the presence of CD34 surface glycoprotein through immunocytochemistry (ICC) and flowcytometry. Initially, these HSCs were induced with activin-A (10ng/ml), retinoic acid (RA) (10ng/ml) and bone morphogenic protein (BMP-7) (2.5ng/ml) for 5days. Transdifferentiation of HSCs to podocytes through intermediate mesoderm was studied with positive selection of Osr1+ cells. Subsequently, thus-obtained Osr1+ cells were induced further with activin-A (10ng/ml), RA (10ng/ml), BMP-7 (2.5ng/ml), EGF (30ng/ml) and bFGF (30ng/ml) for 9days. Distinct cobblestone morphological changes were observed on staining with Leishman's stain. Consequently, differentiated cells were immunopositive for anti-podocin, anti-synaptopodin and anti-GLEPP1 monoclonal antibodies. These cells showed expression of early podocyte markers PAX2 and Wt1 at day 3 followed by day 6 and mature podocyte markers NPHS1, SULT1B1, NPHS2 and Synaptopodin at day 9. Interestingly, on day 9, diminished expression of PAX2 was noted. Differentiated cells showed high tyrosine kinase activity signifying that phosphorylation controls slit diaphragm proteins. Synaptopodin regulates the integrity of cytoskeleton and cell motility of podocytes and this phenomenon was confirmed through scratch assay using agarose molds that showed high cell mobility and migration. These findings establish HSCs as ideal candidates for regenerative therapies of damaged podocytes.

An Approach to In Vitro Manufacturing of Hypertrophic Cartilage Matrix for Bone Repair.

Devitalized hypertrophic cartilage matrix (DCM) is an attractive concept for an off-the-shelf bone graft substitute. Upon implantation, DCM can trigger the natural endochondral ossification process, but only when the hypertrophic cartilage matrix has been reconstituted correctly. In vivo hypertrophic differentiation has been reported for multiple cell types but up-scaling and in vivo devitalization remain a big challenge. To this end, we developed a micro tissue-engineered cartilage (MiTEC) model using the chondrogenic cell line ATDC5. Micro-aggregates of ATDC5 cells (approximately 1000 cells per aggregate) were cultured on a 3% agarose mold consisting of 1585 microwells, each measuring 400 µm in diameter. Chondrogenic differentiation was strongly enhanced using media supplemented with combinations of growth factors e.g., insulin, transforming growth factor beta and dexamethasone. Next, mineralization was induced by supplying the culture medium with beta-glycerophosphate, and finally we boosted the secretion of proangiogenic growth factors using the hypoxia mimetic phenanthroline in the final stage of in vivo culture. Then, ATDC5 aggregates were devitalized by freeze/thawing or sodium dodecyl sulfate treatment before co-culturing with human mesenchymal stromal cells (hMSCs). We observed a strong effect on chondrogenic differentiation of hMSCs. Using this MiTEC model, we were able to not only upscale the production of cartilage to a clinically relevant amount but were also able to vary the cartilage matrix composition in different ways, making MiTEC an ideal model to develop DCM as a bone graft substitute.

Development of a novel folate-modified nanobubbles with improved targeting ability to tumor cells

Conjugation of folate (FOL) to nanobubbles could enhance the selective targeting to tumors expressing high levels of folate receptor (FR). To further improve the selective targeting ability of FOL-modified nanobubbles, a novel FOL-targeted nanobubble ((FOL)2-NB) with increasing FOL content (accomplished by linking two FOL molecules per DSPE-PEG2000 chain) was synthesized, through the methods of mechanical shaking and low-speed centrifugation based on lipid-stabilized perfluoropropane. The bubble size and distribution range were measured by dynamic light scattering (DLS). Enhanced imaging ability was evaluated using a custom-made agarose mold with a clinical US imaging system at mechanical indices of up to 0.12 at a center frequency of 9.0MHz. Targeted ability was also carried out in human breast cancer MCF-7 cells, which over-express the FR, by fluorescence activated cell sorting (FACS) and fluorescence microscopy, respectively. (FOL)2-NB with a particle size of 286.87±22.96nm were successfully prepared, and they exhibited superior contrast imaging effect. FACS and fluorescence microscopy studies showed greater cellular targeting ability in the group of (FOL)2-NB than in their control group of Non-targeted-NB (no FOL targeted nanobubbles) and FOL-NB (one FOL molecule per DSPE-PEG2000 chain). These results suggest that a new type of stronger targeted nanobubble was successfully prepared by increasing the FOL content per DSPE-PEG2000 chain. This novel (FOL)2-NBs are potentially useful for ultrasound molecular imaging and treatment of FR-positive tumors and are worthy for further investigation.

Effects of passage number and post-expansion aggregate culture on tissue engineered, self-assembled neocartilage.

Chondrocyte dedifferentiation presents a major barrier in engineering functional cartilage constructs. To mitigate the effects of dedifferentiation, this study employed a post-expansion aggregate culture step to enhance the chondrogenic phenotype of passaged articular chondrocytes (ACs) before their integration into self-assembled neocartilage constructs. The objective was twofold: (1) to explore how passage number (P2, P3, P4, P5, P6, and P7), with or without aggregate culture, affected construct properties; and (2) to determine the highest passage number that could form neocartilage with functional properties. Juvenile leporine ACs were passaged to P2-P7, with or without aggregate culture, and self-assembled into 5mm discs in non-adhesive agarose molds without using any exogenous scaffolds. Construct biochemical and biomechanical properties were assessed. With aggregate culture, neocartilage constructs had significantly higher collagen content, higher tensile properties, and flatter morphologies. These beneficial effects were most obvious at higher passage numbers. Specifically, collagen content, Young's modulus, and instantaneous compressive modulus in the P7, aggregate group were 53%, 116%, and 178% higher than those in the P7, non-aggregate group. Most interestingly, these extensively passaged P7 ACs (expansion factor of 85,000), which are typically highly dedifferentiated, were able to form constructs with properties similar to or higher than those formed by lower passage number cells. This study not only demonstrated that post-expansion aggregate culture could significantly improve the properties of self-assembled neocartilage, but also that chondrocytes of exceedingly high passage numbers, expanded using the methods in this study, could be used in cartilage engineering applications. This work demonstrated that extensively passaged chondrocytes (up to passage 7 (P7); expansion factor of 85,000) could potentially be used for cartilage tissue engineering applications. Specifically, an aggregate culture step, employed after cell expansion and before cell integration into a neocartilage construct, was shown to enhance the ability of the chondrocytes to form neocartilage with better biochemical and biomechanical properties. The beneficial effects of this aggregate culture step was especially noticeable at the high passage numbers. Most interestingly, P7 chondrocytes, which are typically highly dedifferentiated, were able to form neocartilage with properties similar to or higher than those formed by lower passage number cells. The ability to obtain high chondrocyte yields with an enhanced chondrogenic potential could have a broad, beneficial impact in improving current therapies (e.g., using higher cell seeding densities for repair) or developing new strategies that require high cell numbers, such as a scaffold-free approach in forming engineered cartilage.