Impact of bioactive molecule inclusion in lyophilized silk scaffolds varies betweenin vivoandin vitroassessments

Abstract

Biomaterials can influence the coordinated efforts required to achieve tissue rehabilitation. Sponge-like silk fibroin scaffolds that include bioactive molecules have been shown to influence tissue repair. However, the mechanisms by which scaffold formulations elicit desired in vivo responses is unclear. Here, acellular silk scaffolds consisting of type I collagen, heparin, and/or vascular endothelial growth factor (VEGF) were used to investigate material fabrication and composition parameters that drive scaffold degradation, cell infiltration, and adipose tissue deposition in vivo. In subcutaneous implants, scaffold degradation was assessed, and results show that the percentage of cells infiltrating the scaffold increased when scaffold formulations contained bioactive molecules. To gain further insight, calculated in vitro enzymatic degradation rates increased with higher enzyme concentrations and theoretical cleavage sites. However, the addition of type I collagen and heparin to the scaffold at relevant concentrations did not change degradation rates, compared to silk alone. These in vitro results are contrary to observations in vivo, where bioactive molecules influence local protein deposition, immune cell infiltration rates, and vascularization. Thus, quantitative in vitro and in vivo evaluations aid in determining the mechanisms by which biomaterials influence tissue repair and support intentional biomaterial design for clinical applications.

Silk Fibroin Particles as Carriers in the Development of All-Natural Hemoglobin-Based Oxygen Carriers (HBOCs)

“…Implanted neat silk fibroin materials have shown evidence of innate immune phagocytes infiltration at the site of implantation for up to 2 weeks via analysis of CD68 expression, suggesting only mild and acute inflammation, an important step in the tissue healing cascade. 42,63 Previous studies with silk fibroin particles engineered for IP delivery also show mild inflammatory response by macrophages via the monitoring of TNF-α both pre-and post-translation. 48,64,65 Here we observe limited activation of murine blood derived RAW 264.7 macrophages via the assessment of iNOS protein expression in cultures that are stimulated twice over 72 hours (Figure 6).…”

“…This strategy for passive incorporation and stabilization of bioactive molecules and proteins has been investigated and reported previously for a variety of silk fibroin-based materials that contain β-sheet structures. 42,[54][55][56] However, it is critical to confirm 3 main properties of our particle system with the introduction of hemoglobin: 1) limited change in size or polydispersity of the particles so that the neat silk fibroin particles are able to act as controls for future work 2) The spatial orientation of hemoglobin allow for better understanding of intraparticle homogeneity 3) the amount of incorporated hemoglobin that remains active and able to bind and release oxygen. To assess changes in particle size, morphology, and dispersity, we incorporate a ferric (non-oxygen binding) form of hemoglobin termed methemoglobin (mHb).…”

“…We also seek to understand if stabilization in the nano- or microparticle systems differs from the β-sheet crystalline driven process observed in macro-sized silk materials such as films and scaffolds. 33, 42 Additionally, the most common method of synthesis for silk-based anticancer nanomedicines is organic solvent desolvation, 43 which would potentially cause similar damage to hemoglobin cargo as do harsh liposome synthesis methods. Thus, we look to take advantage of the ability of silk fibroin biopolymers to stabilize protein cargo while leveraging an all-aqueous particle fabrication technique inspired by the synthesis of silk nano-porous films.…”

Silk Fibroin Particles as Carriers in the Development of Hemoglobin‐Based Oxygen Carriers

“…We also seek to understand if stabilization in the nano-or microparticle systems differs from the β-sheet crystalline driven process observed in macro-sized silk materials such as films and scaffolds. [40,49] Additionally, the most common method of synthesis for silk-based anticancer nanomedicines is organic solvent desolvation, [30] which would potentially cause oxidative damage to hemoglobin cargo, as seen with some HBOC synthesis methods, which prompted shifts and eventual optimization of formulations and protocols [30] to achieve high levels of encapsulation and minimal hemoglobin damage.…”

“…B) Following deconvolution of the Amide I peak following published methods, [58] β-sheet structures. [49,[61][62][63] However, it is critical to confirm three main properties of our particle system with the introduction of hemoglobin: 1) limited change in size or polydispersity of the particles so that the neat silk fibroin particles are able to act as controls for future work; 2) the spatial orientation of hemoglobin allow for better understanding of intraparticle homogeneity; and 3) the amount of incorporated hemoglobin that remains active and able to bind and release oxygen. To assess changes in particle size, morphology, and dispersity, we incorporate a ferric (nonoxygen binding) form of hemoglobin termed methemoglobin (mHb).…”