Hix, J. | Search bioRxiv

bioRxiv2024

Device Design and Diagnostic Imaging of Radiopaque 3D Printed Tissue Engineering Scaffolds

3D printed biomaterial implants are revolutionizing personalized medicine for tissue repair, especially in orthopedics. In this study, a radiopaque Bi2O3 doped polycaprolactone (PCL) composite is developed and implemented to enable the use of diagnostic X-ray technologies, especially photon counting X-ray computed tomography (PCCT), for comprehensive in vivo device monitoring. PCL filament with homogeneous Bi2O3 nanoparticle (NP) dispersion (0.8 to 11.7 wt%) are first fabricated. Tissue engineered scaffolds (TES) are then 3D printed with the composite filament, optimizing printing parameters for small feature size and severely overhung geometries. These composite TES are characterized via micro-computed tomography ({micro}CT), tensile testing, and a cytocompatibility study, with Bi2O3 mass fractions as low as 2 wt% providing excellent radiographic distinguishability, improved tensile properties, and equivalent cytocompatibility of neat PCL. The excellent radiographic distinguishability is validated in situ by imaging 4 and 7 wt% TES in a mouse model with {micro}CT, showing excellent agreement with in vitro measurements. Subsequently, CT image-derived swine menisci are 3D printed with composite filament and re-implanted in their corresponding swine legs ex vivo. Re-imaging the swine legs via clinical CT allows facile identification of device location and alignment. Finally, the emergent technology of PCCT unambiguously distinguishes implanted menisci in situ.

bioengineering↗

bioRxiv2020

Ciona Brachyury proximal and distal enhancers have different FGF dose-response relationships

Many genes are regulated by two or more enhancers that drive similar expression patterns. Evolutionary theory suggests that these seemingly redundant enhancers must have functionally important differences. In the simple ascidian chordate Ciona, the transcription factor Brachyury is induced exclusively in the presumptive notochord downstream of lineage specific regulators and FGF-responsive Ets family transcription factors. Here we exploit the ability to finely titrate FGF signaling activity via the MAPK pathway using the MEK inhibitor U0126 to quantify the dependence of transcription driven by different Brachyury reporter constructs on this direct upstream regulator. We find that the more powerful promoter-adjacent proximal enhancer and a weaker distal enhancer have fundamentally different dose-response relationships to MAPK inhibition. The Distal enhancer is more sensitive to MAPK inhibition but shows a less cooperative response, whereas the Proximal enhancer is less sensitive and more cooperative. A longer construct containing both enhancers has a complex dose-response curve that supports the idea that the proximal and distal enhancers are moderately super-additive. We show that the overall expression loss from intermediate doses of U0126 is not only a function of the fraction of cells expressing these reporters, but also involves graded decreases in expression at the single-cell level. Expression of the endogenous gene shows a comparable dose-response relationship to the full length reporter, and we find that different notochord founder cells are differentially sensitive to MAPK inhibition. Together, these results indicate that although the two Brachyury enhancers have qualitatively similar expression patterns, they respond to FGF in quantitatively different ways and act together to drive high levels of Brachyury expression with a characteristic input/output relationship. This indicates that they are fundamentally not redundant genetic elements.

developmental biology↗