RESUMO
Immediate care of skin wounds and burns is essential to repair this mechanical and chemical barrier to infections. Hydrogels have become one of the standard methods for wound care. Here, gelatine-collagen photo-crosslinkable matrixes or hydrogels were manufactured by two-photon polymerization (TPP) or one-photon UV exposure using a Digital Light Processing (DLP) setup. Both techniques are able to construct matrixes from computer-aided design models, which is important for future clinical applications in which wound dressings should be customized. Although TPP can mimic the 3D dermo-epidermal junction with a high spatial resolution (i.e., â¼6 µm3), the manufacturing time was too slow to produce large wound dressings. Therefore, a DLP setup was explored in this study to fabricate large 2D matrixes of several cm2 using the same photo-resist as for TPP, except for the photoinitiator. The fibroblast viability, adherence, and proliferation were analysed in time on both 3D and 2D matrixes in vitro using two-photon microscopy. For both types of matrixes, the adherence and proliferation of fibroblasts (3T3-NIH) were optimal for stiff structures with a Young's modulus of 191 ± 35 kPa compared to softer matrixes of 37 ± 12 kPa. Fibroblast showed complete confluence on Day 14 after seeding on these matrixes, which may create the granulation tissue composed of fibronectin, collagen, and various proteoglycans in the future dermis before repair of the epidermis and disintegrating of their host matrix. For the monitoring of this repair, gelatine-collagen matrixes can easily incorporate bio-optical sensors for the simultaneous monitoring of inflammation processes and wound healing in time.
Assuntos
Gelatina , Pele , Colágeno , Regeneração , HidrogéisRESUMO
The extra cellular matrix plays a major role in the biomechanical properties of tissues that impact cell behavior and fate. It is therefore crucial to mimic these complex cell-matrix interactions in 3D cell cultures. Here, two-photon polymerization is applied to produce gelatin methacryloyl (GelMA) - collagen matrixes that further enable local pO2matrix measurement, when ruthenium complexes are used as photo-activators. The fluorescence intensity of these complexes has a direct and inverse relationship with the local pO2matrix. The 3D structures reached their maximum size in cell culture conditions after 3H with a swelling factor of ~1.5. Their shape and the ruthenium fluorescence intensity of the alveoli walls stayed constant for at least 2 weeks in the absence of cells. They were used in time series to monitor the local pO2matrix adjacent to cancer cells during their division, migration and the formation of a tumor tissue mass. At the presence of these cell activities that consume O2, a significant ~3-fold increase of the ruthenium fluorescence intensity in the alveoli walls was observed. This study demonstrates that online monitoring of the local pO2matrix is possible. The ruthenium complexes provide the bio-optical sensors that are useful for further analysis of cancer and healthy cell energy metabolism in a 3D matrix that better mimics in vivo conditions and migration paths. Unraveling the cancer cell metabolic adaptations in a changing micro-environment will help the development of new therapeutic opportunities. STATEMENT OF SIGNIFICANCE: In 3D cell cultures, monitoring pericellular pO2 is as critical as controlling pH. This facility is currently missing. Here, we take advantage of the direct and inverse relationship between pO2 and the fluorescence intensity of ruthenium complexes to generate stable gelatin-collagen matrixes able to continuously monitoring the pO2 at the pericellular level. The ruthenium complexes, which are photo-activators in the two-photon polymerization of these matrixes, became covalently bind to the collagen fibers. Indeed, local O2 consumption by cancer cells during migration, mitosis and tumor mass formation caused a 3-fold increase of the ruthenium fluorescence. In the future, incorporating ruthenium complexes with other bio-optical sensors will create new drug screening platforms that monitor cell culture parameters at the pericellular level.
