PurposeCollagen scaffolds are used as tissue engineering tool. The manufacturing methods often lack of reproducibility which limits their application to regenerative medicine. We aimed to set a simple and reproducible method for the production of collagen scaffolds for tissue engineering.MethodsA matryoshka system was built comprising a sealed desiccator containing a saturated K2CO3 solution for a 40% relative humidity (RH) regulation, this was placed inside a 40° C shaking incubator. Collagen gels were cast with a 1:1 ratio of Dulbecco’s Modified Eagle’s medium and 0.5 mg/mL acid collagen solution. Three gel volumes were selected: 2.2 mL (1x), 4.4 mL (2x), and 6.6 mL (3x). Collagen gels were inserted into the system and desiccation was performed over 37 days (6–12 mL was desiccated per cycle). Characterization of the surface, ultrastructure, transparency, composition, and biocompatibility of the gels was performed using optical, 3D confocal, and scanning electron microscopy; spectrophotometry; Fourier-transform infrared spectroscopy; and X-Ray diffraction. ResultsOn day 7, collagen membranes exhibited superficial fibrils of 1.3 µm (SD, ± 0.23 µm), whereas on day 37, a highly organized laminar structure was observed within a smooth homogeneous surface. Lamina density and organization and membrane width (3.65 µm [1x], 4.8 µm [2x], and 7.2 µm [3x]) increased with gel volume. Transmittance ranged from 77% to 99% and increased with wavelength at UV–vi. Gels at 1x and 2x exhibited a 99% transmittance peak at the green wavelength. The population of cells cultured on membranes was increased by threefold within 48 h; moreover, the size of cell populations cultured on 1x membranes increased by 12% compared with the control. ConclusionsThe scaffolds produced by the matrioshka system were biocompatible, non-cytotoxic, and optically transparent. These membranes can be tailored for multiple uses by modifying their thickness with the volume of the gel and its desiccation time.

We set a feasible method to produce tailored collagen scaffolds and analyzed its potential for corneal engineering. Collagen-vitrigel membranes (CVM) were produced with a 1:1 ratio of Dulbecco’s Modified Eagle’s medium (DMEM), 1% antibiotics and 8% fetal bovine serum, and 5mg/mL collagen type I. Three volumes of collagen were used: 1X (2.8 L/mm2 of collagen), 2X, and 3X. Vitrification was done at 40% relative humidity (RH), 40° C, and 30 rpm using a matryoshka system set with a shaking-oven and a desiccator with a saturated K2CO3 solution. The CVM was characterized for width, microstructure, transparency, and biocompatibility using NIH3T3 cells. Surgical manipulation was assessed in an ex vivo corneal model. Constructs of corneal endothelial cells (CECs) and 2X-CVM were transplanted into five 18-month-old White New Zealand rabbits. CVM exhibited homogeneous surface and laminar organization. Membrane width increased with gel volume from 3.65µm to 7.2µm. 1X and 2X-CVM exhibited a 99% transmittance. NIH3T3 cells concentration increased 3-fold within 48 h with no significant difference among the 3 CVM (p = 0.323). The 2X-CVM was surgically manipulable. Transplantation of corneal endothelial cells (CECs) seeded over 2X-CVM restored corneal endothelium. The matrioshka system is a feasible method that yields CVM suitable for corneal engineering.