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Enhanced osteoblastogenesis in three-dimensional collagen gels.

ABSTRACT: Growth and differentiation of osteoblasts are often studied in cell cultures. In vivo, however, osteoblasts are embedded within a complex three-dimensional (3D) microenvironment, which bears little relation to standard culture flasks. Our study characterizes osteoblast-like cells cultured in 3D collagen gels and compares them with cells in two-dimensional (2D) cultures. Primary rat osteoblasts and MC3T3-E1 cells were seeded within type I collagen gels, and differentiation was determined by mineral staining and gene expression analysis. Cells growing in 3D gels showed positive mineral staining and induction of osteoblast marker genes earlier than cells growing in 2D. A number of genes, including osteocalcin, bone sialoprotein, alkaline phosphatase and dentin matrix protein 1, were already highly upregulated in 3D cultures 24 h after seeding. The early expression of osteoblast genes was dependent on the 3D structure and was not induced in cells growing on collagen-coated dishes in 2D. Comparison of thymidine incorporation between cells in 3D and 2D cultures treated with agents that induce proliferation-transforming growth factor β, platelet-derived growth factor and lactoferrin-showed a much greater response in 3D gels. Cells in 3D cultures were also much more sensitive to inhibition of proliferation by the protein kinase inhibitor imatinib mesylate. The 3D collagen gels better represent the physiological bone environment and offer a number of technical advantages for the study of osteoblasts in vitro. These studies have additional practical implications as 3D collagen gels are considered as a scaffold material in regenerative medicine for the repair of bone defects.

SUBMITTER: Matthews BG

PROVIDER: S-EPMC4130130 | biostudies-other | 2014

REPOSITORIES: biostudies-other

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Enhanced neurite growth from mammalian neurons in three-dimensional salmon fibrin gels.

Project description:Three-dimensional fibrin matrices have been used as cellular substrates in vitro and as bridging materials for central nervous system repair. Cells can be embedded within fibrin gels since the polymerization process is non-toxic, making fibrin an attractive scaffold for transplanted cells. Most studies have utilized fibrin prepared from human or bovine blood proteins. However, fish fibrin may be well suited for neuronal growth since fish undergo remarkable central nervous system regeneration and molecules implicated in this process are present in fibrin. We assessed the growth of mammalian central nervous system neurons in bovine, human, and salmon fibrin and found that salmon fibrin gels encouraged the greatest degree of neurite (dendrite and axon) growth and were the most resistant to degradation by cellular proteases. The neurite growth-promoting effect was not due to the thrombin used to polymerize the gels nor to any copurifying plasminogen. Copurified fibronectin partially accounted for the effect on neurites, and blockade of fibrinogen/fibrin-binding integrins markedly decreased neurite growth. Anion exchange chromatography revealed different elution profiles for salmon and mammalian fibrinogens. These data demonstrate that salmon fibrin encourages the growth of neurites from mammalian neurons and suggest that salmon fibrin may be a beneficial scaffold for neuronal regrowth after CNS injury.

| S-EPMC1991290 | biostudies-literature

Elastic moduli of collagen gels can be predicted from two-dimensional confocal microscopy.

Project description:We quantitatively compare data obtained from imaging two-dimensional slices of three-dimensional unlabeled and fluorescently labeled collagen gels with confocal reflectance microscopy (CRM) and/or confocal fluorescence microscopy (CFM). Different network structures are obtained by assembling the gels over a range of concentrations at various temperatures. Comparison between CRM and CFM shows that the techniques are not equally sensitive to details of network structure, with CFM displaying higher fidelity in imaging fibers parallel to the optical axis. Comparison of CRM of plain and labeled collagen gels shows that labeling itself induces changes in gel structure, chiefly through inhibition of fibril bundling. Despite these differences, image analyses carried out on two-dimensional CFM and CRM slices of collagen gels reveal identical trends in structural parameters as a function of collagen concentration and gelation temperature. Fibril diameter approximated from either CRM or CFM is in good accord with that determined via electron microscopy. Two-dimensional CRM images are used to show that semiflexible polymer theory can relate network structural properties to elastic modulus successfully. For networks containing bundled fibrils, it is shown that average structural diameter, rather than fibril diameter, is the length scale that sets the magnitude of the gel elastic modulus.

| S-EPMC2756380 | biostudies-literature

Alignment of astrocytes increases neuronal growth in three-dimensional collagen gels and is maintained following plastic compression to form a spinal cord repair conduit.

Project description:After injury to the spinal cord, reactive astrocytes form a glial scar consisting of highly ramified cell processes that constitute a major impediment to repair, partly due to their lack of orientation and guidance for regenerating axons. In some nonmammalian vertebrates, successful central nervous system regeneration is attributed to the alignment of reactive glia, which guide axons across the lesion site. Here, a three-dimensional mammalian cell-seeded collagen gel culture system was used to explore the effect of astrocyte alignment on neuronal growth. Astrocyte alignment was mapped within tethered rectangular gels and was significantly greater at the edge and middle of the gels compared to the control unaligned regions. When neurons were seeded on and within astrocyte gels, neurite length was greatest in the areas of astrocyte alignment. There was no difference in expression of astrocyte reactivity markers between aligned and control areas. Having established the potential utility of astrocyte alignment, the aligned gels were plastic compressed, transforming them into mechanically robust implantable devices. After compression, astrocytes remained viable and aligned and supported neurite outgrowth, yielding a novel method for assembling aligned cellular constructs suitable for tissue engineering and highlighting the importance of astrocyte alignment as a possible future therapeutic intervention for spinal cord repair.

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Glioblastoma behaviors in three-dimensional collagen-hyaluronan composite hydrogels.

Project description:Glioblastoma multiforme (GBM) tumors, which arise from glia in the central nervous system (CNS), are one of the most deadly forms of human cancer with a median survival time of ?1 year. Their high infiltrative capacity makes them extremely difficult to treat, and even with aggressive multimodal clinical therapies, outcomes are dismal. To improve understanding of cell migration in these tumors, three-dimensional (3D) multicomponent composite hydrogels consisting of collagen and hyaluronic acid, or hyaluronan (HA), were developed. Collagen is a component of blood vessels known to be associated with GBM migration; whereas, HA is one of the major components of the native brain extracellular matrix (ECM). We characterized hydrogel microstructural features and utilized these materials to investigate patient tumor-derived, single cell morphology, spreading, and migration in 3D culture. GBM morphology was influenced by collagen type with cells adopting a rounded morphology in collagen-IV versus a spindle-shaped morphology in collagen-I/III. GBM spreading and migration were inversely dependent on HA concentration; with higher concentrations promoting little or no migration. Further, noncancerous astrocytes primarily displayed rounded morphologies at lower concentrations of HA; in contrast to the spindle-shaped (spread) morphologies of GBMs. These results suggest that GBM behaviors are sensitive to ECM mimetic materials in 3D and that these composite hydrogels could be used to develop 3D brain mimetic models for studying migration processes.

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Vinculin facilitates cell invasion into three-dimensional collagen matrices.

Project description:The cytoskeletal protein vinculin contributes to the mechanical link of the contractile actomyosin cytoskeleton to the extracellular matrix (ECM) through integrin receptors. In addition, vinculin modulates the dynamics of cell adhesions and is associated with decreased cell motility on two-dimensional ECM substrates. The effect of vinculin on cell invasion through dense three-dimensional ECM gels is unknown. Here, we report how vinculin expression affects cell invasion into three-dimensional collagen matrices. Cell motility was investigated in vinculin knockout and vinculin expressing wild-type mouse embryonic fibroblasts. Vinculin knockout cells were 2-fold more motile on two-dimensional collagen-coated substrates compared with wild-type cells, but 3-fold less invasive in 2.4 mg/ml three-dimensional collagen matrices. Vinculin knockout cells were softer and remodeled their cytoskeleton more dynamically, which is consistent with their enhanced two-dimensional motility but does not explain their reduced three-dimensional invasiveness. Importantly, vinculin-expressing cells adhered more strongly to collagen and generated 3-fold higher traction forces compared with vinculin knockout cells. Moreover, vinculin-expressing cells were able to migrate into dense (5.8 mg/ml) three-dimensional collagen matrices that were impenetrable for vinculin knockout cells. These findings suggest that vinculin facilitates three-dimensional matrix invasion through up-regulation or enhanced transmission of traction forces that are needed to overcome the steric hindrance of ECMs.

| S-EPMC2857131 | biostudies-literature

A three-dimensional computational model of collagen network mechanics.

Project description:Extracellular matrix (ECM) strongly influences cellular behaviors, including cell proliferation, adhesion, and particularly migration. In cancer, the rigidity of the stromal collagen environment is thought to control tumor aggressiveness, and collagen alignment has been linked to tumor cell invasion. While the mechanical properties of collagen at both the single fiber scale and the bulk gel scale are quite well studied, how the fiber network responds to local stress or deformation, both structurally and mechanically, is poorly understood. This intermediate scale knowledge is important to understanding cell-ECM interactions and is the focus of this study. We have developed a three-dimensional elastic collagen fiber network model (bead-and-spring model) and studied fiber network behaviors for various biophysical conditions: collagen density, crosslinker strength, crosslinker density, and fiber orientation (random vs. prealigned). We found the best-fit crosslinker parameter values using shear simulation tests in a small strain region. Using this calibrated collagen model, we simulated both shear and tensile tests in a large linear strain region for different network geometry conditions. The results suggest that network geometry is a key determinant of the mechanical properties of the fiber network. We further demonstrated how the fiber network structure and mechanics evolves with a local formation, mimicking the effect of pulling by a pseudopod during cell migration. Our computational fiber network model is a step toward a full biomechanical model of cellular behaviors in various ECM conditions.

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Microstructural and mechanical differences between digested collagen-fibrin co-gels and pure collagen and fibrin gels.

Project description:Collagen and fibrin are important extracellular matrix (ECM) components in the body, providing structural integrity to various tissues. These biopolymers are also common scaffolds used in tissue engineering. This study investigated how co-gelation of collagen and fibrin affected the properties of each individual protein network. Collagen-fibrin co-gels were cast and subsequently digested using either plasmin or collagenase; the microstructure and mechanical behavior of the resulting networks were then compared with the respective pure collagen or fibrin gels of the same protein concentration. The morphologies of the collagen networks were further analyzed via three-dimensional network reconstruction from confocal image z-stacks. Both collagen and fibrin exhibited a decrease in mean fiber diameter when formed in co-gels compared with the pure gels. This microstructural change was accompanied by an increased failure strain and decreased tangent modulus for both collagen and fibrin following selective digestion of the co-gels. In addition, analysis of the reconstructed collagen networks indicated the presence of very long fibers and the clustering of fibrils, resulting in very high connectivities for collagen networks formed in co-gels.

| S-EPMC3462241 | biostudies-literature

Genome-wide analysis of control and MMP-14 (MT1-MMP) expressing MCF-7 cells growing in three-dimensional (3D) type I collagen gels versus monolayer cell culture conditions

2014-12-15 | GSE49595 | GEO

Strain-induced alignment in collagen gels.

Project description:Collagen is the most abundant extracellular-network-forming protein in animal biology and is important in both natural and artificial tissues, where it serves as a material of great mechanical versatility. This versatility arises from its almost unique ability to remodel under applied loads into anisotropic and inhomogeneous structures. To explore the origins of this property, we develop a set of analysis tools and a novel experimental setup that probes the mechanical response of fibrous networks in a geometry that mimics a typical deformation profile imposed by cells in vivo. We observe strong fiber alignment and densification as a function of applied strain for both uncrosslinked and crosslinked collagenous networks. This alignment is found to be irreversibly imprinted in uncrosslinked collagen networks, suggesting a simple mechanism for tissue organization at the microscale. However, crosslinked networks display similar fiber alignment and the same geometrical properties as uncrosslinked gels, but with full reversibility. Plasticity is therefore not required to align fibers. On the contrary, our data show that this effect is part of the fundamental non-linear properties of fibrous biological networks.

| S-EPMC2691583 | biostudies-literature

Genome-wide analysis of control and MMP-14 (MT1-MMP) expressing MCF-7 cells growing in three-dimensional (3D) type I collagen gels versus monolayer cell culture conditions

Project description:In order to investigate the impact of MMP-14 (MT1-MMP) and three-dimensional (3D) culture conditions on the transcriptomes of a human breast adenocarcinoma cell line, we performed a microarray analysis from RNAs isolated from MCF-7 cells expressing either an empty vector (CTRL) or human MMP-14 cDNA (MT1) in monolayer (2D) and 3D collagen (3D Col) growth conditions. MCF-7 cells were stably transfected with either an empty vector (pcDNA3.1/Zeo) or human MMP-14 cDNA (pcDNA3.1-MMP-14/Zeo). Cells were grown for 24, 48 and 72 hours in three-dimensional (3D) type I collagen gels or in monolayer culture conditions. Cells were then lysed in TRIzol and total RNA was isolated. For each experimental condition, total RNAs isolated from 4 independant biological replicates were pooled.

2014-12-15 | E-GEOD-49595 | biostudies-arrayexpress

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