Functional characterization of ß2-adrenergic and insulin receptor heteromers.

This study aimed to functionally characterize ß2-adrenergic (ß2AR) and insulin receptor (IR) heteromers in regard to ß-arrestin 2 (ßarr2) recruitment and cAMP signaling and to examine the involvement of the cytoplasmic portion of the IR ß chain in heteromerization with ß2AR. Evidence for ß2AR:IR:ßarr2 complex formation and the specificity of the IR:ßarr2 interaction was first provided by bioinfomatics analysis. Receptor-heteromer investigation technology (HIT) then provided functional evidence of ß2AR:IR heterodimerization by showing isoproterenol-induced but not insulin-induced GFP2-ßarr2 recruitment to the ß2AR:IR complex; the IR:ßarr2 interaction was found to only be constitutive. The constitutive IR:ßarr2 BRET signal (BRETconst) was significantly smaller in cells coexpressing IR-RLuc8 and a GFP2-ßarr2 1-185 mutant lacking the proposed IR binding domain. ß2AR:IR heteromerization also influenced the pharmacological phenotype of ß2AR, i.e., its efficacy in recruiting ßarr2 and activating cAMP signaling. Evidence suggesting involvement of the cytoplasmic portion of the IR ß chain in the interaction with ß2AR was provided by BRET2 saturation and HIT assays using an IR 1-1271 stop mutant lacking the IR C-terminal tail region. For the complex consisting of IR 1-1271-RLuc8:ß2AR-GFP2, saturation was not reached, most likely reflecting random collisions between IR 1-1271 and ß2AR. Furthermore, in the HIT assay, no substantial agonist-induced increase in the BRET2 signal was detected that would be indicative of ßarr2 recruitment to the IR 1-1271:ß2AR heteromer. Complementary 3D visualization of ß2AR:IR provided supporting evidence for stability of the heterotetramer complex and identified amino acid residues involved in ß2AR:IR heteromerization. This article is part of the Special Issue entitled 'Receptor heteromers and their allosteric receptor-receptor interactions'.

Polyester type polyHIPE scaffolds with an interconnected porous structure for cartilage regeneration.

Development of artificial materials for the facilitation of cartilage regeneration remains an important challenge in orthopedic practice. Our study investigates the potential for neocartilage formation within a synthetic polyester scaffold based on the polymerization of high internal phase emulsions. The fabrication of polyHIPE polymer (PHP) was specifically tailored to produce a highly porous (85%) structure with the primary pore size in the range of 50-170 µm for cartilage tissue engineering. The resulting PHP scaffold was proven biocompatible with human articular chondrocytes and viable cells were observed within the materials as evaluated using the Live/Dead assay and histological analysis. Chondrocytes with round nuclei were organized into multicellular layers on the PHP surface and were observed to grow approximately 300 µm into the scaffold interior. The accumulation of collagen type 2 was detected using immunohistochemistry and chondrogenic specific genes were expressed with favorable collagen type 2 to 1 ratio. In addition, PHP samples are biodegradable and their baseline mechanical properties are similar to those of native cartilage, which enhance chondrocyte cell growth and proliferation.

Microcellular open porous monoliths for cell growth by thiol-ene polymerization of low-toxicity monomers in high internal phase emulsions.

Open porous microcellular polymers with high degrees of porosity are prepared from divinyl adipate and pentaerythritol tetrakis(3-mercaptopropionate) by thiol-ene polymerization within high internal phase emulsions. The influence of monomer ratio, droplet phase volume, and emulsion stirring rate on the morphology and mechanical properties of the products is studied. The newly produced material is successfully applied as a scaffold for osteoblastic MC3T3-E1 cells in vitro, showing increased rates of cell growth compared to material prepared by standard methods.

Hierarchically porous materials from layer-by-layer photopolymerization of high internal phase emulsions.

A combination of high internal phase emulsion (HIPE) templating and additive manufacturing technology (AMT) is applied for creating hierarchical porosity within an acrylate and acrylate/thiol-based polymer network. The photopolymerizable formulation is optimized to produce emulsions with a volume fraction of droplet phase greater than 80 vol%. Kinetic stability of the emulsions is sufficient enough to withstand in-mold curing or computer-controlled layer-by-layer stereolithography without phase separation. By including macroscale cellular cavities within the build file, a level of controlled porosity is created simultaneous to the formation of the porous microstructure of the polyHIPE. The hybrid HIPE-AMT technique thus provides hierarchically porous materials with mechanical properties tailored by the addition of thiol chain transfer agent.