Merlin instrument

Morphological evaluation was performed to observe the reconstitution of collagen fibrils at physiological temperature and pH, and the distribution of the inorganic phases into the collagen matrix.
Before printing, bulk samples of Coll/nanoMBG/nanoHA were obtained by pipetting the collagen‐based suspension in a silicon mold, that was subsequently incubated at 37°C for 3 h.
For the analyses, both bulk samples and chemically crosslinked 3D printed scaffolds were lyophilized for 24 h after freezing at −20°C, using a Lyovapor L‐200 freeze‐dryer (Büchi, Switzerland) under vacuum (<0.1 mbar). Cross‐sections of lyophilized samples were sputter‐coated with platinum (7 nm thickness layer) and analyzed through Field‐Emission Scanning Electron Microscopy (FESEM) with a ZEISS MERLIN instrument (Carl Zeiss AG, Oberkochen, Germany).
The chemical composition of the developed biomaterial was then confirmed by means of Fourier‐transform infrared spectroscopy (FTIR) and by using the attenuated total reflection (ATR) mode. The resulting spectrum, in the 4000–650 cm⁻¹ range, were collected by using a Bruker Equinox 55 spectrometer, equipped with MCT cryodetector, at a spectral resolution of 4 cm⁻¹ and accumulation of 32 scans.

Prior to analysis with FE-SEM, samples were frozen at −20 °C for 24 h, and then lyophilized with a Lyovapor L-200 freeze-dryer (Büchi, Cornaredo, Italy) under vacuum (<0.1 mbar) for 24 h. The scaffolds were coated with a 7 nm platinum layer and analyzed with a ZEISS MERLIN instrument (Carl Zeiss AG, Oberkochen, Germany).

Absorption and photoluminescence (PL) spectra were collected on a spectrophotometer UV-3600 (Shimadzu, Kyoto, Japan) and on spectrofluorometers FP-8200 (Jasco, Tokyo, Japan) and FLS920P (Edinburgh Instruments, Livingston, UK), respectively. PL QY was measured with an integrating sphere (Labsphere, North Sutton, NH, US), using a 405 nm diode laser as an excitation source. Fourier transform infrared (FTIR) spectra were collected on an infrared spectrophotometer Tenzor II (Bruker, Billerica, MA, US). Scanning electron microscopy (SEM) images were collected on a Merlin instrument (Zeiss, Oberkochen, Germany). Optical imaging has been performed on a confocal microscope LSM-710 (Zeiss, Oberkochen, Germany) equipped with 20× objective (NA  =  0.4) and a 405 nm diode laser as an excitation source. Time-resolved PL measurements were performed on a confocal microscope MicroTime 100 (PicoQuant, Berlin, Germany) equipped with 100× objective (NA  =  0.95) or 3× objective (NA  =  0.1), and a 405 nm pulsed diode laser. PL decay curves were fitted by a biexponential function: $I\left(t\right)=I_0+{\mathrm{A}}_1{\mathrm{e}}^{-\mathrm{t}/{\mathsf{\tau }}_1}+{\mathrm{A}}_2{\mathrm{e}}^{-\mathrm{t}/{\mathsf{\tau }}_2}$ . The average PL lifetime has been calculated as $\langle \tau \rangle ={{\displaystyle \sum }}^{ }{\mathrm{A}}_i{{\tau }_i}^2/{{\displaystyle \sum }}^{ }{\mathrm{A}}_i{\tau }_i$ .