Properties of crosslinked protein matrices for tissue engineering applications synthesized by multiphoton excitation

Abstract

We demonstrate the fabrication of model scaffolds and extracellular matrices using multiphoton excited photochemistry. This method is three-dimensional in nature and has excellent biocompatibility. Crosslinked matrices were fabricated from the proteins fibrinogen, fibronectin, and concanavalin A using two-photon rose bengal photoactivation and the relatives rates were determined. Immunofluorescence labeling of fibrinogen and fibronectin indicated retention of bioactivity following the multiphoton crosslinking process. Using the fluorescence recovery after photobleaching method, we measured the lateral mobility of fluorescent dyes of different mass and chemistry in order to model the behavior of therapeutic agents and bioactive molecules and found diffusion coefficients within these fabricated structures to be on the order of 10⁻⁹–10⁻¹⁰ cm²/s, or approximately three to four orders of magnitude slower than in free solution. The precise diffusion coefficients can be smoothly tuned by varying the laser exposure during the fabrication of the matrix, which results in both an increase in crosslink density as well as protein concentration in the matrix. Terminal crosslink density is achieved at integrated high exposure dose and the relative fabrication rates were determined for these proteins. For all the proteins, the range of diffusion coefficients between the threshold for fabrication and the terminal limit is correlated with the change in matrix mesh size as determined by Flory-Rehner swelling analysis. Both normal Fickian as well as hindered anomalous diffusion is observed depending on specific molecular interactions of the tracer dyes and protein host. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 71A: 359–368, 2004