Non-invasive longitudinal monitoring of vascularization in tissue-engineered grafts by ultrasound localization microscopy

Abstract

Tissue engineering is a rapidly advancing field aimed at regenerating damaged tissues by combining cells, biomaterials, and bioactive molecules. Adequate vascularization is a critical determinant of successful tissue regeneration, as it supports both the regenerative process and the maintenance of physiological functions in the newly formed tissue. Traditional methods for evaluating vascularization, such as histological and immunohistochemical techniques, are inherently invasive and limited to endpoint analysis. In this study, we innovatively explored the potential of ultrasound localization microscopy (ULM) as a non-invasive, high-resolution, and longitudinal imaging tool for monitoring vascularization in tissue-engineered grafts. Three representative types of tissue-engineered grafts, Hydrogel, Hydrogel with stem cells, and Hydrogel with growth factors were subcutaneously implanted in mice. ULM imaging was performed at multiple time points (7, 14, and 21 days post-implantation) to evaluate vascular growth within the implants dynamically. The vascularization of these grafts was further validated using conventional H&E staining and CD31 immunohistochemical staining. Our findings demonstrated that ULM had a strong correlation (R² = 0.82 and 0.91, respectively) with conventional histological methods, confirming its accuracy as a non-invasive tool for vascular assessment in tissue engineering research.

Statement of significance

Adequate vascularization is fundamental for successful tissue regeneration, but traditional methods like histology are invasive and limited to endpoint analysis. This study innovatively utilizes ultrasound localization microscopy (ULM) as a non-invasive, high-resolution tool for longitudinal monitoring of vascularization in three tissue-engineered grafts: Hydrogel, Hydrogel with stem cells, and Hydrogel with growth factors. ULM showed a strong correlation (R2 = 0.82 and 0.91, respectively) with histological assessments (H&E staining and CD31 staining) across all time points. Notably, this represents the first demonstration of ULM for non-invasive, dynamic tracking of graft vascularization. With its deep tissue penetration and robust reliability, ULM emerges as a promising alternative for evaluating vascularization in tissue regeneration research, enabling continuous, high-precision analysis without tissue excision.