Estrogen Signaling Dictates Musculoskeletal Stem Cell Behavior: Sex Differences in Tissue Repair.

Sexual dimorphisms in humans and other species exist in visually evident features such as body size and less apparent characteristics, including disease prevalence. Current research is adding to a growing understanding of sex differences in stem cell function and response to external stimuli, including sex hormones such as estrogens. These differences are proving significant and directly impact both the understanding of stem cell processes in tissue repair and the clinical implementation of stem cell therapies. Adult stem cells of the musculoskeletal system, including those used for development and repair of muscle, bone, cartilage, fibrocartilage, ligaments, and tendons, are no exception. Both in vitro and in vivo studies have found differences in stem cell number, proliferative and differentiation capabilities, and response to estrogen treatment between males and females of many species. Maintaining the stemness and reducing senescence of adult stem cells is an important topic with implications in regenerative therapy and aging. As such, this review discusses the effect of estrogens on musculoskeletal system stem cell response in multiple species and highlights the research gaps that still need to be addressed. The following evidence from investigations of sex-related phenotypes in adult progenitor and stem cells are pieces to the big puzzle of sex-related effects on aging and disease and critical information for both fundamental tissue repair and regeneration studies and safe and effective clinical use of stem cells. Impact Statement This review summarizes current knowledge of sex differences in and the effects of estrogen treatment on musculoskeletal stem cells in the context of tissue engineering. Specifically, it highlights the impact of sex on musculoskeletal stem cell function and ability to regenerate tissue. Furthermore, it discusses the varying effects of estrogen on stem cell properties, including proliferation and differentiation, important to tissue engineering. This review aims to highlight the potential impact of estrogens and the importance of performing sex comparative studies in the field of tissue engineering.

Surfactant location and internal phase volume fraction dictate emulsion electrospun fiber morphology and modulate drug release and cell response.

Emulsion electrospinning is a versatile technique used to create fibrous meshes for applications in drug delivery and tissue engineering. In this study, the effects of surfactant and increasing internal phase volume fraction on emulsion electrospun fiber morphology were investigated. The fiber diameter, surface topography, internal architecture, mesh hydrophobicity, and fiber volume fraction were all characterized and the resulting effects on model drug release and cell response were determined. Surfactant relocation to the fiber surface resulted in alterations to fiber surface topography and internal morphology, increased rate of water adsorption into the mesh, and reduced burst effects of drug release. Increasing the internal phase volume fraction within the emulsion resulted in minimal change to fiber diameter, surface morphology, fiber volume fraction, and rate of water adsorption illustrating the ability to increase drug loading without affecting fiber properties. Lastly, all meshes promoted cell adhesion and good viability with a trend of increased MTT absorbance from cells on the surfactant and emulsion fibers possibly suggesting that an increase in surface area via smaller fiber diameter and fiber volume fraction increases metabolic activity. Overall, these studies indicate that fiber morphology and mesh hydrophobicity can be tuned by controlling surfactant location within fibers and internal phase volume fraction. Modulating fiber properties within the emulsion electrospun mesh is important to achieve controlled drug release and cell response for tissue engineering applications.

Antibody-Drug Conjugate that Exhibits Synergistic Cytotoxicity with an Endosome-Disruptive Peptide.

Antibody-drug conjugates are an important class of cancer therapeutics. These agents generally bind a specific cell surface receptor, undergo receptor-mediated endocytosis, and enter the endosomal-lysosomal system, where the environment in these organelles facilitates the release of a membrane-permeable cytotoxin. By using a membrane-impermeable cytotoxin, we describe here a method that allows the cytotoxicity of an antibody conjugate to be triggered by co-administration with an endosome-disruptive peptide that exhibits low toxicity. This approach was validated by conjugation of an anionic derivative of the tubulin-binding cytotoxin colchinol methyl ether to lysine residues of the HER2-targeting antibody trastuzumab (Herceptin) via a disulfide. When this antibody binds HER2 on SKBR3 breast cancer cells and undergoes endocytosis, the membrane-impermeable cytotoxin is released, but it becomes trapped in endosomes, resulting in relatively low cytotoxicity (IC50 > 1 µM). However, co-administration with an essentially nontoxic (IC50 > 10 µM) cholesterol-linked endosome-disruptive peptide promotes the release of this small molecule into the cytoplasm, conferring subnanomolar cytotoxic potency (IC50 = 0.11 ± 0.07 nM). Studies of a structurally related fluorophore conjugate revealed that the endosome-disruptive peptide does not substantially enhance cleavage of the disulfide (t 1/2 = 8 ± 2 h) within endosomes, suggesting that the mechanism of endosomal escape involves the efflux of some small molecules without facilitating substantial influx of reduced glutathione.

Targeting Fluorescent Sensors to Endoplasmic Reticulum Membranes Enables Detection of Peroxynitrite During Cellular Phagocytosis.

Peroxynitrite is a highly reactive oxidant derived from superoxide and nitric oxide. In normal vertebrate physiology, some phagocytes deploy this oxidant as a cytotoxin against foreign pathogens. To provide a new approach for detection of endogenous cellular peroxynitrite, we synthesized fluorescent sensors targeted to membranes of the endoplasmic reticulum (ER). The very high surface area of these membranes, approximately 30 times greater than the cellular plasma membrane, was envisioned as a vast intracellular platform for the display of sensors to transient reactive species. By linking an ER-targeted profluorophore to reactive phenols, sensors were designed to be cleaved by peroxynitrite and release a highly fluorescent ER-associated rhodol. Studies of kinetics in aqueous buffer revealed a linear free energy relationship where electron-donating substituents accelerate this reaction. However, in living cells, the efficiency of detection of endogenous cellular peroxynitrite was directly proportional to association with ER membranes. By incorporating a 2,6-dimethylphenol to accelerate the reaction and enhance this subcellular targeting, endogenous peroxynitrite in living RAW 264.7 macrophage cells could be readily detected after addition of antibody-opsonized tentagel microspheres, without additional stimulation, a process undetectable with other known fluorescent sensors. This approach provides uniquely sensitive tools for studies of transient reactive species in living mammalian cells.