Multimode 8 hr scanning probe microscope

To assess the earliest stages of peptide-mediated mineralization at the interface between collagen and co-polymerizable hydroxyapatite-binding peptide (MAHABP), type I collagen model specimens prepared by spin-coating were analyzed using atomic force microscopy. Aqueous collagen solution (type I) from rat tail (Sigma C3867) mixed with 4mg/ml HABP or MAHABP was coated on round glass coverslip (12 mm diameter, 26023, Ted Pella Inc.) with a WS-400E-6NPP-LITE spin coater (Laurell Technologies, North Wales, PA) at speed of 1000 RPM. The resultant thin film was incubated with mineralization solution for 20 or 40 min, and then analyzed using AFM and Raman. The AFM images were obtained with a Multimode 8 HR scanning probe microscope (Bruker Corporation, Camarillo, CA) operated in tapping mode under ambient conditions (24 ± 2°C, 40% ± 5% RH). Tapping mode etched silicon probes (Prod No.: RTESPA-300, Bruker) were used, having a resonant frequency of about 285 KHz. The length and thickness of the probes were 115–135 μm and 38–42 μm, respectively. Images of each sample were recorded with Nanoscope 8.15 software and analyzed with the Nanoscope Analysis 2.0 software. Related, but separate samples of type I collagen model specimens mixed with MAHABP were analyzed using Raman.

A Bruker Multimode 8 HR scanning probe microscope (Bruker Nano Inc., Camarillo, CA, USA) was operated in peak force tapping mode with the capability of Quantitative Nanomechanics (PeakForce-QNM) in air mode conditions (24 ± 2 °C, 40% ± 5% RH). This advanced testing mode was used to examine the topographical and nanomechanical property changes of the collagen-peptide samples both before and after mineralization. Tapping mode etched silicon probes, type RTESPA 525-30 (Bruker Nano Inc., Camarillo, CA, USA) with a resonant frequency of about 518 kHz, were used to acquire images (1 μm × 3 μm and 5 μm × 5 μm) at scan rate of 0.5 Hz with 512 pixel/line resolution. The images of the samples were recorded using NanoScope 8.15 software and analyzed using NanoScope Analysis 2.0 software (Bruker Nano Inc., Camarillo, CA, USA).