Tendon tissue engineering requires the generation of a uniaxially orientated collagen type I matrix with several organization scales that confer mechanical functionality upon the tendon. A combination of factors in a dose- and time-dependent manner, such as growth factors and mechanical environment, may be the key to an in vitro-engineered tendon. To define the progress of tissue development within a scaffold, on-line systems need to be applied to monitor the newly generated matrix. To address this challenge, we designed a new porous chitosan scaffold with microchannels (diameter: 250 microm), which allows primary porcine tenocytes to proliferate in a bundle-like structure. The cell proliferation and extracellular matrix (ECM) production within the microchannels were successfully assessed under sterile conditions using optical coherence tomography (OCT). A semi-quantitative method that calculated the microchannel occupation ratio (the degree of cell proliferation and tissue turnover based on the total backscattered intensity in the microchannels) was developed. We further investigated the effect of different culture conditions on tendon cell matrix formation. Using a perfusion bioreactor, we demonstrated how fluid flow can increase (p < 1e(3)) ECM production within the microchannels significantly more than static culture. Our study illustrates how using a guiding scaffold in combination with the fast and non-destructive assessment of the microstructure using OCT allows discrimination between the parameters affecting the production and the organization of the ECM.