Spectrum bx

UV and IR spectra were obtained employing Perkin-Elmer Lambda 40 and Perkin-Elmer Spectrum BX instruments (PerkinElmer, Inc., Waltham, USA), respectively. VLC grade (Macherey-Nagel, Polygoprep 60-50C18) was used for vacuum liquid chromatography. All organic solvents were distilled prior to use. HPLC was carried out using a Waters system, controlled by Waters Millenium software, consisting of a 600E pump, a 996 PDA, and a 717 plus autosampler (Waters Corporation, Milford, MA, USA). All NMR spectra were recorded on a Bruker Avance 300 and 500 DRX spectrometers (Bruker Corporation, Rheinstetten, Germany). Spectra were referenced to the residual solvent signals with resonances at δ_H/C 2.04/29.8 (acetone-d₆) and δ_H/C 7.26/77.0 (CDCl₃). ESI mass spectra were obtained on an Applied Biosystems/MDS Sciex API 2000 MS spectrometer (Applera Corporation and MDS Inc., Foster, CA, USA). HRESIMS were recorded on a Bruker Daltonik micrOTOF-Q Time-of-Flight mass spectrometer with ESI source, and UPLC-Synapt G2 HDMS mass spectrometer (Bruker CorporationCompany, Rheinstetten, Germany).

Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy was performed on dry wet spun fibres using a Perkin-Elmer Spectrum BX (Waltham, MA, USA) over a range of 800–4000 cm⁻¹ at a resolution of 2 cm⁻¹.
Differential Scanning Calorimetry (DSC) was employed to investigate the thermal denaturation of collagen samples (TA Instruments Thermal Analysis 2000 System and 910 Differential Scanning Calorimeter cell base, New Castle, DE, USA). DSC temperature scans were conducted in a temperature range of 10–100 °C using a 10 °C·min⁻¹ heating rate. Next, 5–10 mg of wet sample (n = 2) was applied in each measurement and two scans were used for each sample formulation. The DSC cell was calibrated using indium and zinc with a 10 °C·min⁻¹ heating rate under 30 cm³·min⁻¹ of nitrogen atmosphere.
Wide-angle X-ray scattering (WAXS) was carried out on dry collagen networks with a Bruker D8 Discover (40 kV, 30 mA, l = 0.154 nm). The detector was set at a distance of 150 mm covering 2θ from 3° to 40°. The collimator was 2.0 mm wide, and the exposure time was 10 s per frame. Collected curves were subtracted from the background (no sample loaded) curve and fitted with polynomial functions (R² > 0.93).

Cells were harvested in ammoniated water (50 mM ammonium bicarbonate, pH 8), lyophilized and analyzed as KBr pellets using Fourier Transformed Infrared (Luppen et al., 2003 (link)). Briefly, 100 mg samples were ground to homogeneity in 1 mg KBr powder using an agate mortar and pestle and pressed into thin pellets. Scans were taken at the rate of 64 cm⁻¹ at 2.0 cm⁻¹ interval using the Perkin Elmer Spectrum BX. Baseline correction, spectrum smoothing, and normalization were done using a Spectrum v5.0.1 (Perkin Elmer Instruments LLC). Overlapping peaks were resolved using second derivative methodology and the curve fitted with a mixed Gaussian and Lorentzian function using PeakFit v4.12. The degree of matrix mineralization was calculated using integrative area of v1, v3 phosphate (1,200–900 cm⁻¹) and amide I (1,720–1,590 cm⁻¹), and the carbonate:phosphate ratio was calculated based on the region of v2 carbonate (890–850 cm⁻¹) (Kato et al., 2001 (link)). The degree of crystal maturity was calculated as the peak area of 1,030:1,110 (Farlay et al., 2010 (link)). Calvaria used as a positive control for well-mineralized bone was obtained from the same mice from which the pulp was isolated.