Local estrogen for nonsurgical recontouring of auricular cartilage.

INTRODUCTION: 5% of children are born with auricular deformities. Permanent recontouring can be achieved through splinting during early infancy. Beyond this time, splinting is ineffective, and patients require surgical correction. Neonatal cartilage malleability is hypothesized to be secondary to retained maternal estrogens, increasing hyaluronic acid concentration. In this article, we evaluate the efficacy of local estrogen treatments for the nonsurgical recontouring of mature auricular cartilage. METHODS: Ears of New Zealand rabbits were folded and splinted and then were randomly assigned to an experimental group, n = 10 (injected estrogen, topical estrogen, saline, or untreated). Treatment ears received injected estrogen or saline twice weekly or topical estrogen daily for 4 weeks. Two weeks post-treatment, splints were removed, and ear angles were measured. Biopsies were taken for histologic and mechanical analysis, and systemic estrogen levels were assayed. RESULTS: Ear angles stabilized by 9 days post-splinting. Topical estrogen led to a significantly smaller resting angle (121.6° ± 13.5°) compared with saline and control (135.9° ± 11.2° and 145.3° ± 13.0°, respectively). Injected estrogen led to the most pronounced angle decrease (64.5° ± 35.3°). Ears injected with estrogen also showed a significant increase in cartilage thickness. Hyaluronic acid concentration was increased in both estrogen treatment groups compared with saline. At 3 weeks post-treatment, there was no significant differences in the elastic modulus of the cartilage or serum estrogen levels among the groups. CONCLUSION: Results show the potential result of local estrogen treatment to achieve a stable nonsurgical remodeling of mature auricular cartilage. Further study is needed to evaluate the molecular mechanism and improve the transdermal estrogen delivery to optimize treatment regimen.

The Combined Influence of Viscoelastic and Adhesive Cues on Fibroblast Spreading and Focal Adhesion Organization.

INTRODUCTION: Tissue fibrosis is characterized by progressive extracellular matrix (ECM) stiffening and loss of viscoelasticity that ultimately impairs organ functionality. Cells bind to the ECM through integrins, where αv integrin engagement in particular has been correlated with fibroblast activation into contractile myofibroblasts that drive fibrosis progression. There is a significant unmet need for in vitro hydrogel systems that deconstruct the complexity of native tissues to better understand the individual and combined effects of stiffness, viscoelasticity, and integrin engagement on fibroblast behavior. METHODS: We developed hyaluronic acid hydrogels with independently tunable cell-instructive properties (stiffness, viscoelasticity, ligand presentation) to address this challenge. Hydrogels with mechanics matching normal or fibrotic lung tissue were synthesized using a combination of covalent crosslinks and supramolecular interactions to tune viscoelasticity. Cell adhesion was mediated through incorporation of either RGD peptide or engineered fibronectin fragments promoting preferential integrin engagement via αvß3 or α5ß1. RESULTS: On fibrosis-mimicking stiff elastic hydrogels, preferential αvß3 engagement promoted increased spreading, actin stress fiber organization, and focal adhesion maturation as indicated by paxillin organization in human lung fibroblasts. In contrast, preferential α5ß1 binding suppressed these metrics. Viscoelasticity, mimicking the mechanics of healthy tissue, largely curtailed fibroblast spreading and focal adhesion organization independent of adhesive ligand type, highlighting its role in reducing fibroblast-activating behaviors. CONCLUSIONS: Together, these results provide new insights into how mechanical and adhesive cues collectively guide disease-relevant cell behaviors. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00672-1.

Click-functionalized hydrogel design for mechanobiology investigations.

The advancement of click-functionalized hydrogels in recent years has coincided with rapid growth in the fields of mechanobiology, tissue engineering, and regenerative medicine. Click chemistries represent a group of reactions that possess high reactivity and specificity, are cytocompatible, and generally proceed under physiologic conditions. Most notably, the high level of tunability afforded by these reactions enables the design of user-controlled and tissue-mimicking hydrogels in which the influence of important physical and biochemical cues on normal and aberrant cellular behaviors can be independently assessed. Several critical tissue properties, including stiffness, viscoelasticity, and biomolecule presentation, are known to regulate cell mechanobiology in the context of development, wound repair, and disease. However, many questions still remain about how the individual and combined effects of these instructive properties regulate the cellular and molecular mechanisms governing physiologic and pathologic processes. In this review, we discuss several click chemistries that have been adopted to design dynamic and instructive hydrogels for mechanobiology investigations. We also chart a path forward for how click hydrogels can help reveal important insights about complex tissue microenvironments.

Spatiotemporal Control of Viscoelasticity in Phototunable Hyaluronic Acid Hydrogels.

Viscoelasticity has emerged as a critical regulator of cell behavior. However, there is an unmet need to develop biomaterials where viscoelasticity can be spatiotemporally controlled to mimic the dynamic and heterogeneous nature of tissue microenvironments. Toward this objective, we developed a modular hyaluronic acid hydrogel combining light-mediated covalent and supramolecular cross-linking to afford spatiotemporal control of network viscoelastic properties. Covalently cross-linked elastic hydrogels or viscoelastic hydrogels combining covalent and supramolecular interactions were fabricated to match healthy and fibrotic liver mechanics. LX-2 human hepatic stellate cells cultured on viscoelastic hydrogels displayed reductions in spreading, actin stress fiber organization, and myocardin-related transcription factor A (MRTF-A) nuclear localization compared to cells on elastic hydrogels. We further demonstrated the dynamic capabilities of our hydrogel system through photo-mediated secondary incorporation of either covalent or supramolecular cross-links to modulate viscoelastic properties. We used photopatterning to create hydrogels with well-controlled patterned regions of stiff elastic mechanics representing fibrotic tissue nodules surrounded by regions of soft viscoelastic hydrogel mimicking healthy tissue. Cells responded to the local mechanics of the patterned substrates with increased spreading in fibrosis-mimicking regions. Together, this work represents an important step forward toward the creation of hydrogel models with spatiotemporal control of both stiffness and viscoelastic cell-instructive cues.

Combined effect of synthetic protein, Mini-B, and cholesterol on a model lung surfactant mixture at the air-water interface.

The overall goal of this work is to study the combined effects of Mini-B, a 34 residue synthetic analog of the lung surfactant protein SP-B, and cholesterol, a neutral lipid, on a model binary lipid mixture containing dipalmitolphosphatidylcholine (DPPC) and palmitoyl-oleoyl-phosphatidylglycerol (POPG), that is often used to mimic the primary phospholipid composition of lung surfactants. Using surface pressure vs. mean molecular area isotherms, fluorescence imaging and analysis of lipid domain size distributions; we report on changes in the structure, function and stability of the model lipid-protein films in the presence and absence of varying composition of cholesterol. Our results indicate that at low cholesterol concentrations, Mini-B can prevent cholesterol's tendency to lower the line tension between lipid domain boundaries, while maintaining Mini-B's ability to cause reversible collapse resulting in the formation of surface associated reservoirs. Our results also show that lowering the line tension between domains can adversely impact monolayer folding mechanisms. We propose that small amounts of cholesterol and synthetic protein Mini-B can together achieve the seemingly opposing requirements of efficient LS: fluid enough to flow at the air-water interface, while being rigid enough to oppose irreversible collapse at ultra-low surface tensions.