Differential Smad2/3 linker phosphorylation is a crosstalk mechanism of Rho/ROCK and canonical TGF-ß3 signaling in tenogenic differentiation.

The transforming growth factor (TGF)-ß3 is a well-known inducer for tenogenic differentiation, signaling via the Smad2/3 pathway. Furthermore, other factors like extracellular matrix or mechanical force can induce tenogenic differentiation and possibly alter the response to TGF-ß3 by signaling via the Rho/ROCK pathway. The aim of this study was to investigate the interplay of Rho/ROCK and TGF-ß3/Smad signaling in tenogenic differentiation, with the Smad2/3 molecule hypothesized as a possible interface. Cultured as monolayers or on collagen I matrices, mesenchymal stromal cells (MSC) were treated with the ROCK inhibitor Y-27632 (10 µM), TGF-ß3 (10 ng/ml) or both combined. Control cells were cultured accordingly, without Y-27632 and/or without TGF-ß3. At different time points, MSC were analyzed by real-time RT-PCR, immunofluorescence, and Western blot. Cultivation of MSC on collagen matrices and ROCK inhibition supported tenogenic differentiation and fostered the effect of TGF-ß3. The phosphorylation of the linker region of Smad2 was reduced by cultivation on collagen matrices, but not by ROCK inhibition. The latter, however, led to increased phosphorylation of the linker region of Smad3. In conclusion, collagen matrices and the Rho/ROCK signaling pathway influence the TGF-ß3/Smad2/3 pathway by regulating different phosphorylation sites of the Smad linker region.

Fibrillar biopolymer-based scaffolds to study macrophage-fibroblast crosstalk in wound repair.

Controlled wound healing requires a temporal and spatial coordination of cellular activities within the surrounding extracellular matrix (ECM). Disruption of cell-cell and cell-matrix communication results in defective repair, like chronic or fibrotic wounds. Activities of macrophages and fibroblasts crucially contribute to the fate of closing wounds. To investigate the influence of the ECM as an active part controlling cellular behavior, coculture models based on fibrillar 3D biopolymers such as collagen have already been successfully used. With well-defined biochemical and biophysical properties such 3D scaffolds enable in vitro studies on cellular processes including infiltration and differentiation in an in vivo like microenvironment. Further, paracrine and autocrine signaling as well as modulation of soluble mediator transport inside the ECM can be modeled using fibrillar 3D scaffolds. Herein, we review the usage of these scaffolds in in vitro coculture models allowing in-depth studies on the crosstalk between macrophages and fibroblasts during different stages of cutaneous wound healing. A more accurate mimicry of the various processes of cellular crosstalk at the different stages of wound healing will contribute to a better understanding of the impact of biochemical and biophysical environmental parameters and help to develop further strategies against diseases such as fibrosis.

Impact of binding mode of low-sulfated hyaluronan to 3D collagen matrices on its osteoinductive effect for human bone marrow stromal cells.

Synthetically sulfated hyaluronan derivatives were shown to facilitate osteogenic differentiation of human bone marrow stromal cells (hBMSC) by application in solution or incorporated in thin collagen-based coatings. In the presented study, using a biomimetic three-dimensional (3D) cell culture model based on fibrillary collagen I (3D Col matrix), we asked on the impact of binding mode of low sulfated hyaluronan (sHA) in terms of adsorptive and covalent binding on osteogenic differentiation of hBMSC. Both binding modes of sHA induced osteogenic differentiation. Although for adsorptive binding of sHA a strong intracellular uptake of sHA was observed, implicating an intracellular mode of action, covalent binding of sHA to the 3D matrix induced also intense osteoinductive effects pointing towards an extracellular mode of action of sHA in osteogenic differentiation. In summary, the results emphasize the relevance of fibrillary 3D Col matrices as a model to study hBMSC differentiation in vitro in a physiological-like environment and that sHA can display dose-dependent osteoinductive effects in dependence on presentation mode in cell culture scaffolds.

Construction of a 3D brain extracellular matrix model to study the interaction between microglia and T cells in co-culture.

Neurodegenerative disorders are characterised by the activation of brain-resident microglia cells and by the infiltration of peripheral T cells. However, their interplay in disease has not been clarified yet. It is difficult to investigate complex cellular dynamics in living animals, and simple two-dimensional (2D) cell culture models do not resemble the soft 3D structure of brain tissue. Therefore, we developed a biomimetic 3D in vitro culture system for co-cultivation of microglia and T cells. As the activation and/or migration of immune cells in the brain might be affected by components of the extracellular matrix, defined 3D fibrillar collagen I-based matrices were constructed and modified with hyaluronan and/or chondroitin sulphate, resembling aspects of brain extracellular matrix. Murine microglia and spleen-derived T cells were cultured alone or in co-culture on the constructed matrices. Microglia exhibited in vivo-like morphology and T cells showed enhanced survival when co-cultured with microglia or to a minor degree in the presence of glia-conditioned medium. The open and porous fibrillar structure of the matrix allowed for cell invasion and direct cell-cell interaction, with stronger invasion of T cells. Both cell types showed no dependence on the matrix modifications. Microglia could be activated on the matrices by lipopolysaccharide resulting in interleukin-6 and tumour necrosis factor-α secretion. The findings herein indicate that biomimetic 3D matrices allow for co-cultivation and activation of primary microglia and T cells and provide useful tools to study their interaction in vitro.

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 ß1 (TGF-ß1 ). 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-ß1 -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.

Remodeling of Three-Dimensional Collagen I Matrices by Human Bone Marrow Stromal Cells during Osteogenic Differentiation In Vitro.

Cell fate is triggered by the characteristics of the surrounding extracellular matrix (ECM) including its composition and topological and mechanical properties. Human bone marrow stromal cells (hBMSC) are known to reside in a niche environment where they are maintained in a quiescent, multipotent state, also controlled by the ECM characteristics. In this in vitro study, three-dimensional (3D) fibrillary collagen I (Col)-based matrices with defined topological and mechanical characteristics were used (pore size of 3-4 µm, fibril diameter of ∼0.7 µm, ∼90 Pa (non-cross-linked), and ∼160 Pa (cross-linked)), mimicking conditions of the environment in the bone marrow. The performance of non-cross-linked and cross-linked scaffolds during osteogenic differentiation of hBMSC in terms of matrix stiffness and proteolytic degradability was investigated. Cell adhesion, morphology, and invasion as well as matrix remodeling were investigated on cross-linked and non-cross-linked Col matrices over 22 days. About 25% of the cells invaded the matrices and showed a spread morphology independent of cross-linking. Cellular proteolytic matrix degradation in terms of a decreased matrix layer thickness was only found for non-cross-linked matrices at constant pore size and fibril diameter. Osteogenic differentiation of hBMSC was examined by alkaline phosphatase staining and enzyme activity (early marker) and calcium phosphate deposition (late marker) and was similarly supported in both scaffolds. Furthermore, both matrices were strongly stiffened by about 10-fold because of high mineralization under osteogenic conditions. In summary, these results emphasize that fibrillary 3D Col matrices are a suitable model to study primary hBMSC behavior in terms of ECM remodeling during osteogenesis at defined in vitro conditions.

Molecular weight specific impact of soluble and immobilized hyaluronan on CD44 expressing melanoma cells in 3D collagen matrices.

Hyaluronan (HA) and its principal receptor CD44 are known to be involved in regulating tumor cell dissemination and metastasis. The direct correlation of CD44-HA interaction on proliferation and invasion of tumor cells in dependence on the molecular weight and the presentation form of HA is not fully understood because of lack of appropriate matrix models. To address this issue, we reconstituted 3D collagen (Coll I) matrices and functionalized them with HA of molecular weight of 30-50kDa (low molecular weight; LMW-HA) and 500-750kDa (high molecular weight; HMW-HA). A post-modification strategy was applied to covalently immobilize HA to reconstituted fibrillar Coll I matrices, resulting in a non-altered Coll I network microstructure and stable immobilization over days. Functionalized Coll I matrices were characterized regarding topological and mechanical characteristics as well as HA amount using confocal laser scanning microscopy, colloidal probe force spectroscopy and quantitative Alcian blue assay, respectively. To elucidate HA dependent tumor cell behavior, BRO melanoma cell lines with and without CD44 receptor expression were used for in vitro cell experiments. We demonstrated that only soluble LMW-HA promoted cell proliferation in a CD44 dependent manner, while HMW-HA and immobilized LMW-HA did not. Furthermore, an enhanced cell invasion was found only for immobilized LMW-HA. Both findings correlated with a very strong and specific adhesive interaction of LMW-HA and CD44+ cells quantified in single cell adhesion measurements using soft colloidal force spectroscopy. Overall, our results introduce an in vitro biomaterials model allowing to test presentation mode and molecular weight specificity of HA in a 3D fibrillar matrix thus mimicking important in vivo features of tumor microenvironments. STATEMENT OF SIGNIFICANCE: Molecular weight and presentation form (bound vs. soluble) of hyaluronan (HA) are intensively discussed as key regulators in tumor progression and inflammation. We introduce 3D fibrillar collagen matrices with defined microstructure and stiffness allowing the presentation of specific molecular weight forms of HA in soluble and bound manner. Mimicking in that way important in vivo features of tumor microenvironments, we found that only low molecular weight HA (LMW-HA) in soluble form promoted proliferation of a melanoma cell line (BRO), while it enhanced cell invasion in bound form. The molecular weight specificity of LMW-HA was verified to be CD44 receptor dependent and was correlated to adhesive ligand-receptor interactions in quantitative colloidal force spectroscopy at single cell level.