A push for environmentally friendly approaches to biomaterials fabrication has emerged from growing conservational concerns in recent years. Different stages in silk fibroin scaffold production, including sodium carbonate (Na₂CO₃)-based degumming and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)-based fabrication, have drawn attention for their associated environmental concerns. Environmentally friendly alternatives have been proposed for each processing stage; however, an integrated green fibroin scaffold approach has not been characterized or used for soft tissue applications. Here, we show that the combination of sodium hydroxide (NaOH) as a substitute degumming agent with the popular “aqueous-based” alternative silk fibroin gelation method yields fibroin scaffolds with comparable properties to traditional Na₂CO₃-degummed aqueous-based scaffolds. The more environmentally friendly scaffolds were found to have comparable protein structure, morphology, compressive modulus, and degradation kinetics, with increased porosity and cell seeding density relative to traditional scaffolds. Human adipose-derived stem cells showed high viability after three days of culture while seeded in each scaffold type, with uniform cell attachment to pore walls. Adipocytes from human whole adipose tissue seeded into scaffolds were found to have similar levels of lipolytic and metabolic function between conditions, in addition to a healthy unilocular morphology. Results indicate that our more environmentally friendly methodology for silk scaffold production is a viable alternative and well suited to soft tissue applications.

Tissue engineering provides a platform for replacing organs, where biomaterials initially restore function and then degrade as new tissue is formed. However, a significant barrier towards designing biodegradable materials is that patients regenerate new tissue at different rates due to complex, interacting, and currently unpredictable factors. When biomaterials persist beyond the initial healing phase, they are plagued with poor outcomes, so the long-term goal of our work is to develop a personalized biomaterial platform where biomaterial degradation can be non-invasively adjusted using focused ultrasound (FUS) following routine monitoring of how patients are healing. Based on our prior work that indicated FUS induces degradation in silk scaffolds in vitro, and recent work that indicates that FUS can be used to trigger increased degradation behavior in silk scaffolds in vivo, we further investigated the safety and efficacy of this technique by studying the effects of FUS in different regenerative environments. Cy5-labeled silk fibroin scaffolds were implanted either subcutaneously or intramuscularly in the erector spinae muscle of young adult (2-6 months old), middle-aged (8-14 months old), and old-aged (18+ months old) BALB/c mice, and sites were treated with FUS for 5 consecutive days for either 5 or 10 minutes daily, compared to untreated control sites. In vivo fluorescent imaging data from young and middle-aged mice indicated that intramuscular sites treated with 10 minutes of daily FUS experienced the largest degree of scaffold degradation compared to any other site by the termination endpoint. Endpoint histology samples stained with hematoxylin and eosin indicated that cell ingrowth was not inhibited by FUS in aging mice, and TUNEL assays showed that cell apoptosis was not induced via FUS in aging mice. These findings indicate that FUS is safe and effective for triggering scaffold degradation at both subcutaneous and intramuscular sites in aging murine populations.