Tissue engineering and regenerative medicine aim to produce artificial tissues or whole organs for both clinical applications and drug testing, disease models, and cell based biosensors . Three-dimensional (3D) polymeric scaffolds are utilized in tissue engineering to provide biomechanical support for the seeded cells until they are organized into a functional tissue . Studies of cell migration are important for understanding a variety of physiological and pathological processes in tissue regeneration . Researchers have extensively studied the mechanisms and regulation of cell migration in two-dimensional (2D) cell-culture models, where migration is predominantly a function of adhesion and de-adhesion events and where there are no spatial barriers to the advancing cell body . However, discrepancies between the behavior of cells in culture (2D) and in vivo (3D) indicate that it is important to use 3D models to better represent the microenvironment of living tissues with respect to dimensionality, architecture and cell polarity. Unlike cells migrating in 2D, cells migrating in 3D need to overcome the physical resistance provided by the matrix, change shape and morphology, or enzymatically degrade matrix components . Optical techniques can be employed for monitoring cell migration processes in3D scaffolds. In particular, two-photon microscopy (TPM) can be used for monitoring the migration process of living cells also at considerable depths under the scaffold surface. In the present work, the cell migration process of alive chondrocytes labelled with vital PKH67 probe in 3D collagen-based scaffold has been monitored at various times from the seeding process. The results show that TPM can evidence the presence of living cells in different spatial regions whose localizations depend on the position and time elapsed from the seeding.
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