Three-dimensional Collagen Research Articles

Acquired contractile ability in human endometrial stromal cells by passive loading of cyclic tensile stretch

The uterus plays an important and unique role during pregnancy and is a dynamic organ subjected to mechanical stimuli. It has been reported that infertility occurs when the peristalsis is prevented, although its mechanisms remain unknown. In this study, we found that mechanical strain mimicking the peristaltic motion of the uterine smooth muscle layer enabled the endometrial stromal cells to acquire contractility. In order to mimic the peristalsis induced by uterine smooth muscle cells, cyclic tensile stretch was applied to human endometrial stromal cells. The results showed that the strained cells exerted greater contractility in three-dimensional collagen gels in the presence of oxytocin, due to up-regulated alpha-smooth muscle actin expression via the cAMP signaling pathway. These in vitro findings underscore the plasticity of the endometrial stromal cell phenotype and suggest the possibility of acquired contractility by these cells in vivo and its potential contribution to uterine contractile activity. This phenomenon may be a typical example of how a tissue passively acquires new contractile functions under mechanical stimulation from a neighboring tissue, enabling it to support the adjacent tissue’s functions.

Distinct differences in hypoxic responses between human oral mucosa and skin fibroblasts in a 3D collagen matrix.

The differences between oral mucosa and skin wound healing involving hypoxic responses of fibroblasts are poorly elucidated. In this study, we aimed to study the different hypoxic responses between oral and skin fibroblasts embedded in a three-dimensional (3D) collagen matrix to address the early stage of wound healing. Primary oral mucosa fibroblasts (OMFs) obtained from the retromolar area and skin fibroblasts (SFs) obtained from the abdomen were cultured in the 3D 'floating model' under either 21%, 5% or 1% O2 for 2days. Cell viability under hypoxia was higher in the OMFs than in the SFs. Collagen gel contraction was suppressed under hypoxic conditions in both fibroblasts, consistent with the reduction of alpha smooth muscle actin expression, except for SFs under 1% O2. Subsequently, their gene expression profiles between 21 and 1% O2 concentrations were compared via microarray technology, and the expression profiles of the extracellular matrix (ECM)-associated proteins, including matrix metalloproteinases and collagens, were evaluated. The OMFs were more susceptible to 1% O2, and more of their genes were downregulated than the SFs'. Although the production and expression levels of ECM-associated proteins in both fibroblasts diminished under hypoxia, those levels in OMFs were significantly higher than those in SFs. In the case of single origin OMFs and SFs, our findings suggest that OMFs possess a higher baseline production capacity of several ECM-associated proteins than SFs, except type III collagen. The intrinsic hypoxic responses of OMFs may be attributed to a more favourable wound healing in oral mucosa.

Collective forces of tumor spheroids in three-dimensional biopolymer networks.

We describe a method for quantifying the contractile forces that tumor spheroids collectively exert on highly nonlinear three-dimensional collagen networks. While three-dimensional traction force microscopy for single cells in a nonlinear matrix is computationally complex due to the variable cell shape, here we exploit the spherical symmetry of tumor spheroids to derive a scale-invariant relationship between spheroid contractility and the surrounding matrix deformations. This relationship allows us to directly translate the magnitude of matrix deformations to the total contractility of arbitrarily sized spheroids. We show that our method is accurate up to strains of 50% and remains valid even for irregularly shaped tissue samples when considering only the deformations in the far field. Finally, we demonstrate that collective forces of tumor spheroids reflect the contractility of individual cells for up to 1 hr after seeding, while collective forces on longer timescales are guided by mechanical feedback from the extracellular matrix.

Nasal Chondrocyte-Derived Soluble Factors Affect Chondrogenesis of Cocultured Mesenchymal Stem Cells.

To investigate the effect of soluble factors released from human nasal chondrocytes (NCs) on cocultured human bone marrow mesenchymal stem cells (MSCs) and NC tissue-engineered constructs. Cartilage engineered from pure NCs on a three-dimensional (3D) porous collagen scaffold was cultured indirectly in a Transwell system with cartilage engineered from a direct coculture of human bone marrow-derived MSCs and NCs on a 3D porous collagen scaffold. The soluble factors were measured in the conditioned media from the different chambers of the Transwell system. Engineered cartilage from cocultures exposed to the pure NC construct exhibited reduced chondrogenic potential relative to control constructs, shown by reduced extracellular matrix deposition and increased expression of hypertrophic markers. Analysis of the soluble factors within the conditioned media showed an increase in inflammatory cytokines in the coculture chamber exposed to the pure NC construct. Principal component analysis revealed that the majority of the data variance could be explained by proinflammatory factors and hypertrophic chondrogenesis. In conclusion, our data suggest that inflammatory cytokines derived from NCs reduce the chondrogenic potential of coculture engineered cartilage through the induction of hypertrophic chondrogenesis. Impact statement The use of engineered cartilage from cocultured nasal chondrocytes (NCs) and mesenchymal stem cells for nasal cartilage reconstruction may be problematic. Our data suggest that the soluble factors from surrounding native NCs in the cartilage to be fixed can compromise the quality of the engineered cartilage if used in reconstructive surgery.

Collagen Fibril Density Modulates Macrophage Activation and Cellular Functions during Tissue Repair.

Monocytes circulate in the bloodstream, extravasate into the tissue and differentiate into specific macrophage phenotypes to fulfill the immunological needs of tissues. During the tissue repair process, tissue density transits from loose to dense tissue. However, little is known on how changes in tissue density affects macrophage activation and their cellular functions. In this work, monocytic cell line THP-1 cells were embedded in three-dimensional (3D) collagen matrices with different fibril density and were then differentiated into uncommitted macrophages (MPMA) using phorbol-12-myristate-13-acetate (PMA). MPMA macrophages were subsequently activated into pro-inflammatory macrophages (MLPS/IFNγ) and anti-inflammatory macrophages (MIL-4/IL-13) using lipopolysaccharide and interferon-gamma (IFNγ), and interleukin 4 (IL-4) and IL-13, respectively. Although analysis of cell surface markers, on both gene and protein levels, was inconclusive, cytokine secretion profiles, however, demonstrated differences in macrophage phenotype. In the presence of differentiation activators, MLPS/IFNγ secreted high amounts of IL-1β and tumor necrosis factor alpha (TNFα), while M0PMA secreted similar cytokines to MIL-4/IL-13, but low IL-8. After removing the activators and further culture for 3 days in fresh cell culture media, the secretion of IL-6 was found in high concentrations by MIL-4/IL-13, followed by MLPS/IFNγ and MPMA. Interestingly, the secretion of cytokines is enhanced with an increase of fibril density. Through the investigation of macrophage-associated functions during tissue repair, we demonstrated that M1LPS/IFNγ has the potential to enhance monocyte infiltration into tissue, while MIL-4/IL-13 supported fibroblast differentiation into myofibroblasts via transforming growth factor beta 1 (TGF-β1) in dependence of fibril density, suggesting a M2a-like phenotype. Overall, our results suggest that collagen fibril density can modulate macrophage response to favor tissue functions. Understanding of immune response in such complex 3D microenvironments will contribute to the novel therapeutic strategies for improving tissue repair, as well as guidance of the design of immune-modulated materials.

Aberrant Migratory Behavior of Immune Cells in Recurrent Autoimmune Uveitis in Horses.

The participating signals and structures that enable primary immune cells migrating within dense tissues are not completely revealed until now. Especially in autoimmune diseases, mostly unknown mechanisms facilitate autoreactive immune cells to migrate to endogenous tissues, infiltrating and harming organ-specific structures. In order to gain deeper insights into the migratory behavior of primary autoreactive immune cells, we examined peripheral blood-derived lymphocytes (PBLs) of horses with equine recurrent uveitis (ERU), a spontaneous animal model for autoimmune uveitis in humans. In this study, we used a three-dimensional collagen I hydrogel matrix and monitored live-cell migration of primary lymphocytes as a reaction to different chemoattractants such as fetal calf serum (FCS), cytokines interleukin-4 (IL-4), and interferon-γ (IFN-γ), and a specific uveitis autoantigen, cellular retinaldehyde binding protein (CRALBP). Through these experiments, we uncovered distinct differences between PBLs from ERU cases and PBLs from healthy animals, with significantly higher cell motility, cell speed, and straightness during migration of PBLs from ERU horses. Furthermore, we emphasized the significance of expression levels and cellular localization of septin 7, a membrane-interacting protein with decreased abundance in PBLs of autoimmune cases. To underline the importance of septin 7 expression changes and the possible contribution to migratory behavior in autoreactive immune cells, we used forchlorfenuron (FCF) as a reversible inhibitor of septin structures. FCF-treated cells showed more directed migration through dense tissue and revealed aberrant septin 7 and F-actin structures along with different protein distribution and translocalization of the latter, uncovered by immunochemistry. Hence, we propose that septin 7 and interacting molecules play a pivotal role in the organization and regulation of cell shaping and migration. With our findings, we contribute to gaining deeper insights into the migratory behavior and septin 7-dependent cytoskeletal reorganization of immune cells in organ-specific autoimmune diseases.

Microstructural densification and alignment by aspiration-ejection influence cancer cell interactions with three-dimensional collagen networks.

Extracellular matrix microstructure and mechanics are crucial to breast cancer progression and invasion into surrounding tissues. The peritumor collagen network is often dense and aligned, features which in vitro models lack. Aspiration of collagen hydrogels led to densification and alignment of microstructure surrounding embedded cancer cells. Two metastasis-derived breast cancer cell lines, MDA-MB-231 and MCF-7, were cultured in initially 4 mg/ml collagen gels for 3 days after aspiration, as well as in unaspirated control hydrogels. Videomicroscopy during aspiration, and at 0, 1, and 3 days after aspiration, epifluorescence microscopy of phalloidin-stained F-actin cytoskeleton, histological sections, and soluble metabolic byproducts from constructs were collected to characterize effects on the embedded cell morphology, the collagen network microstructure, and proliferation. Breast cancer cells remained viable after aspiration-ejection, proliferating slightly less than in unaspirated gels. Furthermore, MDA-MB-231 cells appear to partially relax the collagen network and lose alignment 3 days after aspiration. Aspiration-ejection generated aligned, compact collagen network microstructure with immediate cell co-orientation and higher cell number density apparently through purely physical means, though cell-collagen contact guidance and network remodeling influence cell organization and collagen network microstructure during subsequent culture. This study establishes a platform to determine the effects of collagen density and alignment on cancer cell behavior, with translational potential for anticancer drug screening in a biomimetic three-dimensional matrix microenvironment, or implantation in preclinical models.

Generation of a three-dimensional collagen scaffold-based model of the human endometrium.

The endometrium is the secretory lining of the uterus that undergoes dynamic changes throughout the menstrual cycle in preparation for implantation and a pregnancy. Recently, endometrial organoids (EO) were established to study the glandular epithelium. We have built upon this advance and developed a multi-cellular model containing both endometrial stromal and epithelial cells. We use porous collagen scaffolds produced with controlled lyophilization to direct cellular organization, integrating organoids with primary isolates of stromal cells. The internal pore structure of the scaffold was optimized for stromal cell culture in a systematic study, finding an optimal average pore size of 101 µm. EO seeded organize to form a luminal-like epithelial layer, on the surface of the scaffold. The cells polarize with their apical surface carrying microvilli and cilia that face the pore cavities and their basal surface attaching to the scaffold with the formation of extracellular matrix proteins. Both cell types are hormone responsive on the scaffold, with hormone stimulation resulting in epithelial differentiation and stromal decidualization.

Loss of smooth muscle α-actin effects on mechanosensing and cell-matrix adhesions.

Mutations in ACTA2, encoding smooth muscle α-actin, are a frequent cause of heritable thoracic aortic aneurysm and dissections. These mutations are associated with impaired vascular smooth muscle cell function, which leads to decreased ability of the cell to sense matrix-mediated mechanical stimuli. This study investigates how loss of smooth muscle α-actin affects cytoskeletal tension development and cell adhesion using smooth muscle cells explanted from aorta of mice lacking smooth muscle α-actin. We tested the hypothesis that reduced vascular smooth muscle contractility due to a loss of smooth muscle α-actin decreases cellular mechanosensing by dysregulating cell adhesion to the matrix. Assessment of functional mechanical properties of the aorta by stress relaxation measurements in thoracic aortic rings suggested two functional regimes for Acta2−/−mice. Lower stress relaxation was recorded in aortic rings from Acta2−/−mice at tensions below 10 mN compared with wild type, likely driven by cytoskeletal-dependent contractility. However, no differences were recorded between the two groups above the 10 mN threshold, since at higher tension the matrix-dependent contractility may be predominant. In addition, our results showed that at any given level of stretch, transmural pressure is lower in aortic rings from Acta2−/−mice than wild type mice. In addition, a three-dimensional collagen matrix contractility assay showed that collagen pellets containing Acta2−/−smooth muscle cells contracted less than the pellets containing the wild type cells. Moreover, second harmonic generation non-linear microscopy revealed that Acta2−/−cells locally remodeled the collagen matrix fibers to a lesser extent than wild type cells. Quantification of protein fluorescence measurements in cells also showed that in absence of smooth muscle α-actin, there is a compensatory increase in smooth muscle γ-actin. Moreover, specific integrin recruitment at cell–matrix adhesions was reduced in Acta2−/−cells. Thus, our findings suggest that Acta2−/−cells are unable to generate external forces to remodel the matrix due to reduced contractility and interaction with the matrix.Impact statementThoracic aneurysm formation is characterized by progressive enlargement of the ascending aorta, which predisposes the aorta to acute aortic dissection that can lead to sudden death. SMCs in the aorta play an integral role in regulating vessel wall contractility and matrix deposition in the medial layer. Recent studies show that mutations in genes associated with actomyosin apparatus reduce SMC contractility, increasing susceptibility to TAAD. Single-cell experiments enable discrete measurements of transient microscopic events that may be masked by a macroscopic average tissue behavior. Biophysical methods combined with microscopy techniques aid in understanding the specific roles of adhesion and cytoskeletal proteins in regulating SMC mechanosensing when SMα-actin is disrupted. Our findings suggest that Acta2−/−cells have increased SMγ-actin and decreased integrin recruitment at cell–matrix adhesion, hence a synthetic phenotype with reduced cellular mechanosensing.

Requirement for the collagen receptor Endo180 in collagen gel contraction mediated by corneal fibroblasts

The interaction of keratocytes with extracellular matrix components plays an important role in the maintenance of corneal transparency and shape as well as in the healing of corneal wounds. In particular, the interaction of these cells with collagen and cell-mediated collagen contraction contribute to wound closure. Endo180 is a receptor for collagen that mediates its cellular internalization. We have now examined the role of Endo180 in collagen contraction mediated by corneal fibroblasts (activated keratocytes). Antibodies to Endo180 inhibited the contractile activity of mouse corneal fibroblasts embedded in a three-dimensional collagen gel and cultured in the presence of serum, with this effect being both concentration and time dependent and essentially complete at an antibody concentration of 0.2 μg/ml. Whereas corneal fibroblasts cultured in a collagen gel manifested a flattened morphology with prominent stress fibers under control conditions, they showed a spindlelike shape with few stress fibers in the presence of antibodies to Endo180. Antibodies to Endo180 had no effect on the expression of α–smooth muscle actin or the extent of collagen degradation in collagen gel cultures of corneal fibroblasts. Immunohistofluorescence analysis did not detect the expression of Endo180 in the unwounded mouse cornea. However, Endo180 expression was detected in keratocytes migrating into the wound area at 3 days after a corneal incisional injury. Together, our results suggest that Endo180 is required for the contraction of collagen matrix mediated by corneal fibroblasts and that its expression in these cells may contribute to the healing of corneal stromal wounds.

Treatment of miller type I and II gingival recession defects using three-dimensional porcine collagen matrix with coronally advanced flap: A randomized clinical split-mouth trial (a 1-year follow-up)

The main goal of periodontal plastic surgery is obtaining complete root coverage (CRC) and an optimal appearance. The aim of this study was to evaluate the effectiveness of a three-dimensional porcine collagen matrix (PCM) with coronally advanced flap (CAF) in treating of Miller type I and II gingival recession (GR). Twenty patients were enrolled in this study, presenting 40 Miller type I and II GR. Patients were randomized into test group (PCM + CAF) and control group [connective tissue graft (CTG + CAF)]. Clinical parameters such as recession depth (RD), probing depth, clinical attachment level (CAL), and width of keratinized gingiva (WKG) were evaluated at baseline and 12 months later. Root coverage percentage (RC%) and CRC were assessed at 12 months post surgically. Statistical analysis was performed using independent t-test for intergroup comparison. Statistical significance was set at 0.05. The mean RD at 12 months was 0.20 mm for the test group and 0.12 mm for the control group, whereas the mean RC% was 94.22% for PCM + CAF and 96.48% for CTG + CAF. CRC was higher in CTG + CAF with 80%. CAL gain was 2.05 and 2.07 mm in the test and control sites, respectively. The gain of WKG was 1.35 and 1.30 mm in the test and control sites, respectively. Patient esthetic satisfaction at 12 months post surgically in both groups was equivalent. Within the limits of this study, using of PCM + CAF in treating GR is a successful and effective treatment option and could serve as an alternative to CTGs.

Three-Dimensional Co-culture of Primary Human Osteocytes and Mature Human Osteoclasts in Collagen Gels.

Osteoclasts are pivotal cells for bone remodeling and their activity is coordinated by osteocytes that reside inside the bone matrix. In vitro co-cultures of osteocytes and osteoclasts are therefore advantageous to analyze the crosstalk between these cell species. In this study, primary osteocytes were isolated from human bone in a multistep isolation process and embedded into three-dimensional collagen gels. Mature human osteoclasts were generated by differentiation of human peripheral blood mononuclear cells (PBMCs). Different surfaces were tested for osteoclast formation: suspension dishes, collagen gels, and normal tissue culture polystyrene. After detachment from the surfaces, osteoclasts showed typical morphology and gene expression of osteoclast markers. Osteoclasts that were differentiated on collagen exhibited the highest osteoclast marker expression. Cocultivation of mature osteoclasts with osteocytes was performed in a transwell system, with osteocytes, embedded in collagen gels at the apical side and osteoclasts on the basal side of a porous polyethylen terephtalate membrane, which allowed the separate gene expression analysis for osteocytes and osteoclasts. After 7 days of co-culture both cell species showed their typical morphology, which is multinucleated giant cells for osteoclasts and star-shaped cells with dendritic extensions for osteocytes. Furthermore, osteoclast markers tartrate-resistant acid phosphatase, carbonic anhydrase II, and cathepsin K were detected both on gene expression and protein level in single and co-cultures. Osteocytes showed gene expression of typical osteocyte markers E11, sclerostin, dentin matrix protein 1, osteocalcin, and receptor activator of nuclear factor-κ ligand both in single and co-culture. Impact statement This study is the first to establish an in vitro bone model that contains both primary human osteocytes and primary human osteoclasts. Previous studies applied rodent osteocyte cell lines to examine the influence of osteocytes on osteoclast function. This model mimics the clinical situation better since osteocytes are postmitotic cells whose function might be different in primary state compared with a proliferating cell line. Furthermore, the co-culture model can be the basis for in vitro triple culture models involving osteoblasts as the third bone cell species.

3D Scaffold-Based Macrophage Fibroblast Coculture Model Reveals IL-10 Dependence of Wound Resolution Phase.

Persistent inflammation and impaired repair in dermal wound healing are frequently associated with cell-cell and cell-matrix miscommunication. A direct coculture model of primary human myofibroblasts (MyoFB) and M-CSF-differentiated macrophages (M-Mɸ) in fibrillar three-dimensional Collagen I (Coll I) matrices is developed to study intercellular interactions. The coculture experiments reveal the number of M-Mɸ regulated MyoFB dedifferentiation in a dose-dependent manner. The amount of MyoFB decreases in dependence of the number of cocultured M-Mɸ, even in the presence of MyoFB-inducing transforming growth factor β1 (TGF-β1 ). Gene expression analysis of matrix proteins (collagen I, collagen III, ED-A-fibronectin) confirms the results of an altered MyoFB phenotype. Additionally, M-Mɸ is shown to be the main source of secreted cytokine interleukin-10 (IL-10), which is suggested to affect MyoFB dedifferentiation. These findings indicate a paracrine impact of IL-10 secretion by M-Mɸ on the MyoFB differentiation status counteracting the TGF-β1 -driven MyoFB activation. Hence, the in vitro coculture model simulates physiological situations during wound resolution and underlines the importance of paracrine IL-10 signals by M-Mɸ. In sum, the 3D Coll I-based matrices with a MyoFB-M-Mɸ coculture form a highly relevant biomimetic model of late stages of wound healing.

Osteogenic cells form mineralized particles, a few μm in size, in a 3D collagen gel culture.

Osteogenic cells form mineralized matrices in vitro, as well as in vivo. The formation and shape of the mineralized matrices are highly regulated by the cells. In vitro formation of mineralized matrices by osteogenic cells can be a model for in vivo osteogenesis. In this study, using a three-dimensional (3D) collagen gel culture system, we developed a new in vitro model for the formation of mineralized particles, a few µm in size, by the osteogenic cells. Human osteosarcoma (HOS) cells formed spherical mineralized matrices (about 12 µm) at approximately 7 days when cultured with β-glycerophosphate (β-GP)-containing culture media on 2D tissue culture plates. Alternately, when they were cultured in a 3D collagen gel containing β-GP, they formed mineralized particles with about 1.7 µm in the gel at approximately 3 days. Calcium precipitation in the gel was evaluated by measuring the gel turbidity. This type of mineralization of HOS cells, which formed mineralized particles inside the gel, was also observed in a peptide-based hydrogel culture. The mineralized particles were completely diminished by inhibiting the activity of Pit-1, phosphate cotransporter, of the HOS cells. When mouse osteoblast-like MC3T3-E1 cells, which form large and flat mineralized matrices in 2D osteogenic conditions at approximately 3 weeks of culture, were cultured in a 3D collagen gel, they also formed mineralized particles in the gel, similar to those in HOS cells, at approximately 18 days. Thus, osteogenic cells cultured in the 3D collagen gel form mineralized particles over a shorter period, and the mineralization could be easily determined by gel turbidity. This 3D gel culture system of osteogenic cells acts as a useful model for cells forming particle-type mineralized matrices, and we assume that the mineralized particles in the 3D hydrogel are calcospherulites, which are derived from matrix vesicles secreted by osteogenic cells.

Extent of Cell Confinement in Microtracks Affects Speed and Results in Differential Matrix Strains

During metastasis, cancer cells navigate through a spatially heterogeneous extracellular matrix (ECM). Physical properties of ECM, including the degree of confinement, influence cell migration behavior. Here, utilizing in vitro three-dimensional collagen microtracks, we demonstrate that cell-ECM interactions, specifically the degree of spatial confinement, regulate migratory behavior. We found that cells migrate faster when they are fully confined, contacting all four walls (top, bottom, and two sides) of a collagen microtrack, compared with cells that are partially confined, contacting less than four walls. When fully confined, cells exhibit fewer but larger vinculin-containing adhesions and create greater strains in the surrounding matrix directed toward the cell body. In contrast, partially confined cells develop a more elongated morphology with smaller but significantly more vinculin-containing adhesions and displace the surrounding matrix less than fully confined cells. The resulting effect of increasing cell contractility via Rho activation is dependent on the number of walls with which the cell is in contact. Although matrix strains increase in both fully and partially confined cells, cells that are partially confined increase speed, whereas those in full confinement decrease speed. Together, these results suggest that the degree of cell-ECM contact during confined migration is a key determinant of speed, morphology, and cell-generated substrate strains during motility, and these factors may work in tandem to facilitate metastatic cell migration.

The Effect of Stichopus chloronotus Aqueous Extract on Human Osteoarthritis Articular Chondrocytes in Three-Dimensional Collagen Type I Hydrogel in vitro

Autologous chondrocyte-seeded scaffolds have proved to be one of the most promising alternative therapies for articular cartilage defects. However, the chondrocytes have specific nutritional requirements and risk of dedifferentiation during in vitro expansion. Stichopus chloronotus aqueous extract (SCAE) was investigated as a medium supplement for three-dimensional (3D) collagen type I hydrogel scaffold seeded with chondrocytes to determine whether SCAE is capable of maintaining phenotype and sustaining extracellular matrix synthesis and deposition. Human osteoarthritis articular chondrocytes were isolated from the knee joint cartilage of patients underwent total knee replacement surgery. Human osteoarthritis articular chondrocytes were encapsulated in collagen type I hydrogel and cultured in basic medium with 0, 0.1 and 0.2% of SCAE. The chondrocytes in 3D culture were evaluated by means cell morphology and proliferation, quantitative phenotypic expression of collagen type I, II, aggrecan core protein and sox-9. H&E, toluidine blue staining and sulfated glycosaminoglycan (sGAG) production were analyzed after 7 days in culture. Chondrocytes cultured in 3D with various SCAE concentration appeared with polygonal morphology maintaining their chondrocytes characteristic. SCAE supplementation promoted chondrocytes proliferation and the ability of the cells to express gene encoding collagen type I decreased, whereas their ability to express collagen type II, aggrecan core protein and sox9 increased as compared to control. The cartilaginous matrices were positively stained toluidine blue concomitant with higher sGAG accumulation in SCAE-supplemented culture medium. This study shown that SCAE may be beneficial for in vitro development of 3D chondrocytes culture for use in cartilage tissue engineering therapies.

Bioactive glass ions for in vitro osteogenesis and microvascularization in gellan gum-collagen hydrogels.

Lack of bone grafts appeals for bone augmentation solutions. We aimed at osteogenic differentiation of human adipose stem cells (hASCs) and microvascularization in coculture with human umbilical vein endothelial cells (HUVECs) embedded in three-dimensional (3D) gellan gum (GG) and collagen type I (COL) hydrogel mixture. We compared endothelial growth medium-2 (EGM-2) and bioactive glass extract-based endothelial and osteogenic medium (BaG EM-OM) for vascularized bone-like graft development in vitro. Cell viability, cell number, and osteogenic and endothelial gene expression were analyzed. Mineralized hydroxyapatite residues, immunocytochemical staining of endothelial marker CD31 production and late osteogenic marker osteocalcin were imaged. With both media, good cell viability was observed within 3D hydrogel. EGM-2 condition induced significantly higher cell number compared to BaG EM-OM condition at both 7 and 14 days. Interestingly, both media supported osteogenic as well as endothelial marker gene expression. Moreover, formation of reticulated cellular structures was observed in both EGM-2 and BaG EM-OM conditions. However, hydroxyapatite mineralization and strong osteocalcin staining were detected only in BaG EM-OM condition. Importantly, strong production of CD31 and elongated tube-like structures were apparent in EGM-2 culture alone. In conclusion, we demonstrated efficient hASC osteogenic differentiation and microvessel-like network formation in coculture with HUVECs.

Cancer Cells Sense Fibers by Coiling on them in a Curvature-Dependent Manner.

SummaryMetastatic cancer cells sense the complex and heterogeneous fibrous extracellular matrix (ECM) by formation of protrusions, and our knowledge of how cells physically recognize these fibers remains in its infancy. Here, using suspended ECM-mimicking isodiameter fibers ranging from 135 to 1,000 nm, we show that metastatic breast cancer cells sense fiber diameters differentially by coiling (wrapping-around) on them in a curvature-dependent manner, whereas non-tumorigenic cells exhibit diminished coiling. We report that coiling occurs at the tip of growing protrusions and the coil width and coiling rate increase in a curvature-dependent manner, but time to maximum coil width occurs biphasically. Interestingly, bundles of 135-nm diameter fibers recover coiling width and rate on 1,000-nm-diameter fibers. Coiling also coincides with curvature-dependent persistent and ballistic transport of endogenous granules inside the protrusions. Altogether, our results lay the groundwork to link biophysical sensing with biological signaling to quantitate pro- and anti-invasive fibrous environments.Video

Synthesis and secretome release by human bone marrow mesenchymal stem cell spheroids within three-dimensional collagen hydrogels: Integrating experiments and modelling.

Myocardial infarction results in loss of cardiac cell types, inflammation, extracellular matrix (ECM) degradation, and fibrotic scar. Transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) is being explored as they could differentiate into cardiomyocyte-like cells, integrate into host tissue, and enhance resident cell activity. The ability of these cells to restore lost ECM, remodel the inflammatory scar tissue, and repair the injured myocardium remains unexplored. We here elucidated the synthesis and deposition of ECM (e.g., elastin, sulfated glycosaminoglycans, hyaluronan, collagen type III, laminin, fibrillin, lysyl oxidase, and nitric oxide synthases), matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), and other secretome (cytokines, chemokines, and growth factors) in adult human BM-MSC spheroid cultures within three-dimensional collagen gels. The roles of species-specific type I collagen and 5-azacytadine were assessed over a 28-day period. Results revealed that human collagen (but not rat-derived) suppressed MSC proliferation and survival, and MSCs synthesized and released a variety of ECM proteins and secretome over the 28 days. Matrix deposition is at least an order of magnitude lower than their release levels at every time point, most possibly due to elevated MMP levels and interleukins with a concomitant decrease in TIMPs. Matrix synthesis over the 28-day period was fitted to a competitive inhibition form of Michaelis-Menten kinetics, and the production and decay rates of ECM, MMPs, and TIMPs, along with the kinetic model parameters quantified. Such an integrated experimental and modelling approach would help elucidate the critical roles of various parameters (e.g., cell encapsulation and delivery vehicles) in stem cell-based transplantation therapies.

Depletion of Ras Suppressor-1 (RSU-1) promotes cell invasion of breast cancer cells through a compensatory upregulation of a truncated isoform

Extracellular matrix (ECM)-adhesion proteins and actin cytoskeleton are pivotal in cancer cell invasion. Ras Suppressor-1 (RSU-1), a cell-ECM adhesion protein that interacts with PINCH-1, thus being connected to Integrin Linked Kinase (ILK), alpha-parvin (PARVA), and actin cytoskeleton, is up-regulated in metastatic breast cancer (BC) samples. Apart from the originally-identified gene (RSU-1L), an alternatively-spliced isoform (RSU-1-X1) has been reported. We used non-invasive MCF-7 cells, expressing only RSU-1L, and highly invasive MDA-MB-231-LM2 expressing both isoforms and generated stable shRNA-transduced cells lacking RSU-1L, while the truncated RSU-1-X1 isoform was depleted by siRNA-mediated silencing. RSU-1L depletion in MCF-7 cells resulted in complete abrogation of tumor spheroid invasion in three-dimensional collagen gels, whereas it promoted MDA-MB-231-LM2 invasion, through a compensatory upregulation of RSU-1-X1. When RSU-1-X1 was also eliminated, RSU-1L-depletion-induced migration and invasion were drastically reduced being accompanied by reduced urokinase plasminogen activator expression. Protein expression analysis in 23 human BC samples corroborated our findings showing RSU-1L to be upregulated and RSU-1-X1 downregulated in metastatic samples. We demonstrate for the first time, that both RSU-1 isoforms promote invasion in vitro while RSU-1L elimination induces RSU-1-X1 upregulation to compensate for the loss. Hence, we propose that both isoforms should be blocked to effectively eliminate metastasis.