Fibril bending stiffness of 3D collagen matrices instructs spreading and clustering of invasive and non-invasive breast cancer cells.

Extracellular matrix stiffening of breast tissues has been clinically correlated with malignant transformation and poor prognosis. An increase of collagen fibril diameter and lysyl-oxidase mediated crosslinking has been observed in advanced tumor stages. Many current reports suggest that the local mechanical properties of single fibrillar components dominantly regulate cancer cell behavior. Here, we demonstrate by an independent control of fibril diameter and intrafibrillar crosslinking of three-dimensional (3D) collagen matrices that fibril bending stiffness instructs cell behavior of invasive and non-invasive breast cancer cells. Two types of collagen matrices with fibril diameter of either 650 nm or 800 nm at a similar pore size of 10 µm were reconstituted and further modified with the zero-length crosslinker 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (EDC) at concentrations of 0, 20, 100 and 500 mM. This approach yields two sets of collagen matrices with overlapping variation of matrix elasticity. With these matrices we could prove the common assumption that matrix elasticity of collagen networks is bending dominated with a linear dependence on fibril bending stiffness. We derive that the measured variation of matrix elasticity is directly correlated to the variation of fibril bending stiffness, being independently controlled either by fibril diameter or by intrafibrillar crosslinking. We use these defined matrices to demonstrate that the adjustment of fibril bending stiffness allows to instruct the behavior of two different breast cancer cell lines, invasive MDA-MB-231 (human breast carcinoma) and non-invasive MCF-7 cells (human breast adenocarcinoma). Invasiveness and spreading of invasive MDA-MB-231 cells as well as clustering of non-invasive MCF-7 cells is thereby investigated over a broad parameter range. Our results demonstrate and quantify the direct dependence of cancer cell phenotypes on the matrix mechanical properties on the scale of single fibrils.

Fibril growth kinetics link buffer conditions and topology of 3D collagen I networks.

Three-dimensional fibrillar networks reconstituted from collagen I are widely used as biomimetic scaffolds for in vitro and in vivo cell studies. Various physicochemical parameters of buffer conditions for in vitro fibril formation are well known, including pH-value, ion concentrations and temperature. However, there is a lack of a detailed understanding of reconstituting well-defined 3D network topologies, which is required to mimic specific properties of the native extracellular matrix. We screened a wide range of relevant physicochemical buffer conditions and characterized the topology of the reconstituted 3D networks in terms of mean pore size and fibril diameter. A congruent analysis of fibril formation kinetics by turbidimetry revealed the adjustment of the lateral growth phase of fibrils by buffer conditions to be key in the determination of pore size and fibril diameter of the networks. Although the kinetics of nucleation and linear growth phase were affected by buffer conditions as well, network topology was independent of those two growth phases. Overall, the results of our study provide necessary insights into how to engineer 3D collagen matrices with an independent control over topology parameters, in order to mimic in vivo tissues in in vitro experiments and tissue engineering applications. STATEMENT OF SIGNIFICANCE: The study reports a comprehensive analysis of physicochemical conditions of buffer solutions to reconstitute defined 3D collagen I matrices. By a combined analysis of network topology, i.e., pore size and fibril diameter, and the kinetics of fibril formation we can reveal the dependence of 3D network topology on buffer conditions, such as pH-value, phosphate concentration and sodium chloride content. With those results we are now able to provide engineering strategies to independently tune the topology parameters of widely used 3D collagen scaffolds based on the buffer conditions. By that, we enable the straightforward mimicking of extracellular matrices of in vivo tissues for in vitro cell culture experiments and tissue engineering applications.

Instructing Human Macrophage Polarization by Stiffness and Glycosaminoglycan Functionalization in 3D Collagen Networks.

Dynamic alterations of composition and mechanics of the extracellular matrix are suggested to modulate cellular behavior including plasticity of macrophages (MPhs) during wound healing. In this study, engineered 3D fibrillar matrices based on naturally occurring biopolymers (collagen I, glycosaminoglycans (GAGs)) are used to mimic matrix stiffening as well as modification by sulfated and nonsulfated GAGs at different stages of wound healing. Human MPhs are found to sensitively respond to these microenvironmental cues in terms of polarization toward proinflammatory or wound healing phenotypes over 6 days in vitro. MPhs exhibit a wound healing phenotype in stiffer matrices as determined by protein and gene expression of relevant cytokines (IL10, IL12, and TNFα). Presence of sulfated and nonsulfated GAGs inhibits this polarization effect. Furthermore, control experiments on 2D matrices stress the relevance of using stiffness-controlled 3D matrices, as MPhs show a reciprocal polarization behavior depending on GAG presence. Hence, the results indicate a strong influence of dimensionality, stiffness, and GAG presence of the biomaterial scaffold on MPh polarization and emphasize the need for matrices closely mimicking the 3D in vivo context with a variable stiffness and GAG composition in in vitro studies.

Surface modification of copolymerized films from three-armed biodegradable macromers - An analytical platform for modified tissue engineering scaffolds.

The concept of macromers allows for a broad adjustment of biomaterial properties by macromer chemistry or copolymerization. Copolymerization strategies can also be used to introduce reactive sites for subsequent surface modification. Control over surface features enables adjustment of cellular reactions with regard to site and object of implantation. We designed macromer-derived polymer films which function as non-implantable analytical substrates for the investigation of surface properties of equally composed scaffolds for bone tissue engineering. To this end, a toolbox of nine different biodegradable, three-armed macromers was thermally cross-copolymerized with poly(ethylene glycol)-methacrylate (PEG-MA) to films. Subsequent activation of PEG-hydroxyl groups with succinic anhydride and N-hydroxysuccinimid allowed for covalent surface modification. We quantified the capacity to immobilize analytes of low (amino-functionalized fluorescent dye, Fcad, and RGD-peptides) and high (alkaline phosphatase, ALP) molecular weight. Fcad grafting level was controlled by macromer chemistry, content and molecular weight of PEG-MA, but also the solvent used for film synthesis. Fcad molar amount per surface area was twentyfive times higher on high-swelling compared to low-swelling films, but differences became smaller when large ALP (appr. 2:1) were employed. Similarly, small differences were observed on RGD peptide functionalized films that were investigated by cell adhesion studies. Presentation of PEG-derivatives on surfaces was visualized by atomic force microscopy (AFM) which unraveled composition-dependent domain formation influencing fluorescent dye immobilization. Surface wetting characteristics were investigated via static water contact angle. We conclude that macromer ethoxylation and lactic acid content determined film swelling, PEG domain formation and eventually efficiency of surface decoration. STATEMENT OF SIGNIFICANCE: Surfaces of implantable biomaterials are the site of interaction with a host tissue. Accordingly, modifications in the composition of the surface will determine cellular response towards the material which is crucial for the success of innovations and control of tissue regeneration. We employed a macromer approach which is most flexible for the design of biomaterials with a broad spectrum of physicochemical characteristics. For ideal analytical accessibility of the material platform, we cross-copolymerized films on solid supports. Films allowed for the covalent immobilization of fluorescent labels, peptides and enzymes and thorough analytical characterization revealed that macromer hydrophilicity is the most relevant design parameter for surface analyte presentation in these materials. All analytical results were combined in a model describing PEG linker domain formation and ligand presentation.

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.

Glycosaminoglycan functionalization of mechanically and topologically defined collagen I matrices.

Collagen I and glycosaminoglycans (GAGs) are major components of the extracellular matrix in mammals and widely used for in vitro cell culture matrices. While composition, network microstructure and mechanics of these matrices sensitively determine cell fate, they are hard to adjust independently during matrix reconstitution. We report on a sequential preparation procedure of collagen I matrices, which allows a defined adjustment of network topology and mechanics in combination with GAG functionalization. Collagen I solution concentrations of 1.5 to 7 mg ml-1 allowed to vary topology (pore size) and elasticity of resulting networks with Young's moduli of 5 to 220 Pa. Zero-length crosslinking using carbodiimide chemistry increased Young's modulus 3 to 5 times without changing network topology. An optional covalent binding of hyaluronan and synthetically sulfated hyaluronan to the preformed matrices led to topologically unaffected networks with a stable functionalization with ∼30 µg GAG per mg collagen. While sulfated GAGs were stably attached to collagen I networks via physisorption or covalent binding at neutral and acidic conditions, non-sulfated hyaluronan required acidic conditions and covalent binding for stable attachment. In conclusion, this approach provides options to independently adjust biophysical and biochemical parameters of collagen I networks for in vitro studies.

Topologically defined composites of collagen types I and V as in vitro cell culture scaffolds.

Cell fate is known to be triggered by cues from the extracellular matrix, including its chemical, biological and physical characteristics. Specifically, mechanical and topological properties are increasingly recognized as important signals. The aim of this work was to provide an easily accessible biomimetic in vitro platform of topologically defined collagen I matrices to dissect cell behaviour under various conditions in vitro. We reconstituted covalently bound layers of three-dimensional (3-D) networks of collagen type I and collagen type V with a defined network topology. A new erosion algorithm enabled us to analyse the mean pore diameter and fibril content, while the mean fibril diameter was examined by an autocorrelation method. Different concentrations and ratios of collagen I and V resulted in pore diameters from 2.4 to 4.5µm and fibril diameters from 0.6 to 0.8µm. A comparison of telopeptide intact collagen I to telopeptide deficient collagen I revealed obvious differences in network structure. The good correlation of the topological data to measurements of network stiffness as well as invasion of human dermal fibroblasts proves that the topological analysis provides meaningful measures of the functional characteristics of the reconstituted 3-D collagen matrices.