In situ ultrasound imaging of silk hydrogel degradation and neovascularization.

Ultrasound (US) is a useful technique to monitor morphological and functional changes of biomaterial implants without sacrificing the animal. Contrast-enhanced ultrasound (CEUS) along with two-dimensional (2D) US were used to characterize the biodegradation and neovascularization of silk protein (8 wt%) hydrogel implants in rats. Cylinder-shaped silk hydrogel plugs were implanted into the space between the hind limb thigh muscles in Wistar rats (n = 6). The increase of echogenicity in 2D US revealed tissue-ingrowth-accompanied gel degradation over 18 weeks. The shape and size of the implanted gels remained qualitatively unchanged until week 15, as confirmed by Bland and Altman analysis and visualization of retrieved samples. Using CEUS, neovascularization was monitored by the presence of microbubbles in the gel area, and the dynamic vascularization process was indicated by the contrast enhancement values, which showed a relatively low level (< 5 dB) during weeks 1-8 and significantly increased levels (around 20 dB at week 15 and > 35 dB at week 18), suggesting that major vascularization had occurred in the gel implants by this time point. Histological and scanning electron microscopic analysis of explants revealed time-dependent increases in the pore size of the gel matrix, the presence of endothelial and red blood cells and the number of blood vessels in the gel implants, indicating that degradation and vascularization did occur in silk gel implants during the time period. The present study demonstrates the use of US imaging for monitoring of in vivo degradation and vascularization of silk implants in a non-destructive way.