Kloxin, A. M. | Search bioRxiv

bioRxiv2020

Multiscale Invasion Assay for Probing Macrophage Response to Bacteria

The immune system is a complex network of various cellular components that must differentiate between pathogenic bacteria and the commensal bacteria of the human microbiome, where misrecognition is linked to inflammatory disorders. Fragments of bacterial cell wall peptidoglycan bind to pattern recognition receptors within macrophages, leading to immune activation. To study this complex process, an approach for three-dimensional (3D) culture of human macrophages and their invasion with relevant bacteria in a well-defined hydrogel-based synthetic matrix inspired by the gut was established. Workflows were developed for monocyte encapsulation and differentiation into macrophages in 3D culture with high viability. Bacteria invaded into macrophages permitted in situ peptidoglycan labeling. Macrophages exhibited biologically-relevant cytokine release in response to bacteria. This multi-dimensional bacteria-macrophage co-culture system will prove useful in future studies to observe bacterial fragment production and localization in the cell at the carbohydrate level for insights into how our immune system properly senses bacteria. TOC Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/385617v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@45cdf1org.highwire.dtl.DTLVardef@d3c3d3org.highwire.dtl.DTLVardef@dd74f2org.highwire.dtl.DTLVardef@128d1da_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Success Criteria for Preclinical Testing of Cell-Instructive Hydrogels for Tendon Regeneration

Tendon injuries are difficult to heal in part because intrinsic tendon healing, which is dominated by scar tissue formation, does not effectively regenerate the native structure and function of healthy tendon. Further, many current treatment strategies also fall short of producing regenerated tendon with the native properties of healthy tendon. There is increasing interest in the use of cell-instructive strategies to limit the intrinsic fibrotic response following injury and improve the regenerative capacity of tendon in vivo. We have established multi-functional, cell-instructive hydrogels for treating injured tendon that afford tunable control over the biomechanical, biochemical, and structural properties of the cell microenvironments. Specifically, we incorporated integrin-binding domains (RGDS) and assembled multi-functional collagen mimetic peptides (mfCMPs) that enable cell adhesion and elongation of stem cells within synthetic hydrogels of designed biomechanical properties and evaluated these materials using targeted success criteria developed for testing in mechanically-demanding environments like tendon healing. The in vitro and in situ success criteria were determined based on systematic reviews of the most commonly reported outcome measures of hydrogels for tendon repair and established standards for testing of biomaterials. We then showed, using validation experiments, that multi-functional and synthetic hydrogels meet these criteria. Specifically, these hydrogels have mechanical properties comparable to developing tendon; are non-cytotoxic both in 2D bolus exposure (hydrogel components) and 3D encapsulation (full hydrogel); are formed, retained, and visualized within tendon defects over time (two-weeks); and provide mechanical support to tendon defects at the time of injection and in situ formation. Ultimately, the in vitro and in situ success criteria evaluated in this study were designed for preclinical research to rigorously test the potential to achieve successful tendon repair prior to in vivo testing and indicate the promise of multi-functional and synthetic hydrogels for continued translation. IMPACT STATEMENTTendon healing results in a weak scar that forms due to poor cell-mediated repair of the injured tissue. Treatments that tailor the instructions experienced by cells during healing afford opportunities to regenerate the healthy tendon. Engineered cell-instructive cues, including the biomechanical, biochemical, and structural properties of the cell microenvironment, within multi-functional synthetic hydrogels are promising therapeutic strategies for tissue regeneration. In this paper, the preclinical efficacy of multi-functional synthetic hydrogels for tendon repair is tested against rigorous in vitro and in situ success criteria. This study indicates the promise for continued preclinical translation of synthetic hydrogels for tissue regeneration.

bioengineering↗