Adherent cells are known to respond to physical characteristics of their surrounding microenvironment, adapting their cytoskeleton and initiating signaling cascades specific to the type of cue encountered. Scaffolds mimicking native biophysical cues have proven to differentiate stem cells towards tissue-specific lineages and to maintain the phenotype of somatic cells for longer periods of time in culture. Biomaterial-based tendon implants are designed to withstand high physiological loads but often lack the appropriate biochemical, biophysical and biological structure to drive tendon regeneration by populating cells. The objective of this study is to use tendon main component, collagen type I, to create scaffolds that reproduce tendon natural anisotropy and rigidity, in an effort to engineer functional tendon tissue with native organization and strength, able to maintain tenocyte phenotype and to differentiate stem cells towards the tenogenic lineage. Porcine collagen type I in solution was treated with one of the following cross-linkers: glutaraldehyde, genipin or 4-arm polyethylene glycol (4SP). The resulting mixture was poured on micro-grooved (2×2×2 um) or planar PDMS moulds and air-dried to obtain 5 mg/ml collagen films. Surface topography and elastic modulus were analyzed using SEM/AFM and rheometry, respectively. Human tendon cells were cultured on the micro-grooved/planar scaffolds for up to 10 days. Cell morphology, collagen III and tenascin C expression were analyzed by immunocytochemistry. Among the different cross-linkers used, only the treatment with 4SP resulted in scaffolds with a recognizable micro-grooved surface topography. Precise control over the micro-grooved topography and the rigidity of the scaffolds was achieved by cross-linking the collagen with varying concentrations of 4SP (0, 0.5, 1 and 1.5mM) at low pH and temperature. The elastic modulus of the scaffolds cross-linked with 4SP (0.5mM) matched the values previously reported to induce tenogenic differentiation in stem cells (50–90 kPa). Approximately eighty percent of the human tendon cells cultured on the micro-grooved collagen films aligned in the direction of the anisotropy for 10 days in culture, mimicking the alignment of tenocytes in the native tissue. Cell nuclei morphology, known to play a central role in the process of mechanotransduction, was significantly more elongated for the tenocytes cultured on the micro-grooved scaffolds after 4 days in culture for all the 4SP concentrations. Synthesis, deposition and alignment of collagen III and tenascin C, two important tenogenic markers, were up regulated selectively on the micro-grooved and rigid scaffolds after 10 days in culture, respectively. These results highlight the synergistic effect of matrix rigidity and cell alignment on tenogenic cell lineage commitment. Collectively, this study provides new insights into how collagen can be modulated to create scaffolds with precise imprinted topographies and controlled rigidities.

Collagen scaffolds are generally characterized by their random fibre distribution and weak mechanical properties, which makes them unsuitable as substitutes for highly anisotropic tissues such as cornea or tendon. Recently, we developed a technique to create collagen type I scaffolds with well-defined anisotropic micro-patterns. Porcine collagen was mixed with PBS10X, NaOH and one of the following cross-linkers: glutaraldehyde (GTA), genipin and 4-arm polyethylene glycol (4SP). The resulting mixture was casted on micro-grooved (2×2×2 μm) polydimethylsiloxane (PDMS) moulds and allowed to dry in a laminar flow hood to obtain 5mg/ml collagen films. Different pH, temperatures (Tº), and cross-linker concentrations were tested in the process. Collagen gelation kinetics was analysed with rheometry and surface topography was assessed with scanning electron microscopy (SEM). Human bone marrow stem cells (HBMSCs) were seeded on the films and cell alignment was analysed by rhodamine/phalloidin staining and imaged with fluorescence microscopy. From all three cross-linkers tested, only 4SP cross-linked scaffolds showed a well-defined micro-grooved pattern. Increasing pH and Tº on 4SP-treated collagen decreased gelation time, which resulted in complete inhibition of the pattern, suggesting that an initial low viscous solution is required for a correct PDMS pattern infiltration. A wide range of 4SP concentrations (0.5, 1, 1.5 mM) maintained the well-defined topography on the films, opening the door to future fine-tuning of the stiffness sensed by cells. hBMSCs seeded on top of the scaffolds aligned along the pattern for 14 days in culture. Collectively, this data highlights the potential of these collagen scaffolds as tendon substitutes.