Schreiber, Areonna C.

AbstractDegenerative spinal pathology is a widespread medical issue, and spine fusion surgeries are frequently performed. In this study, we fabricated an injectable bioactive click chemistry polymer cement for use in spinal fusion and bone regrowth. Taking advantages of the bioorthogonal click reaction, this cement can be crosslinked by itself eliminating the addition of a toxic initiator or catalyst, nor any external energy sources like UV light or heat. Furthermore, nano‐hydroxyapatite (nHA) and microspheres carrying recombinant human bone morphogenetic protein‐2 (rhBMP‐2) and recombinant human vascular endothelial growth factor (rhVEGF) were used to make the cement bioactive for vascular induction and osteointegration. After implantation into a rabbit posterolateral spinal fusion (PLF) model, the cement showed excellent induction of new bone formation and bridging bone, achieving results comparable to autograft control. This is largely due to the osteogenic properties of nano‐hydroxyapatite (nHA) and the released rhBMP‐2 and rhVEGF growth factors. Since the availability of autograft sources is limited in clinical settings, this injectable bioactive click chemistry cement may be a promising alternative for spine fusion applications in addressing various spinal conditions.

Abstract3D stem cell spheroids have immense potential for various tissue engineering applications. However, current spheroid fabrication techniques encounter cell viability issues due to limited oxygen access for cells trapped within the core, as well as nonspecific differentiation issues due to the complicated environment following transplantation. In this study, functional 3D spheroids are developed using mesenchymal stem cells with 2D hetero‐nanostructures (HNSs) composed of single‐stranded DNA (ssDNA) binding carbon nanotubes (sdCNTs) and gelatin‐bind black phosphorus nanosheets (gBPNSs). An osteogenic molecule, dexamethasone (DEX), is further loaded to fabricate an sdCNTgBP‐DEX HNS. This approach aims to establish a multifunctional cell‐inductive 3D spheroid with improved oxygen transportation through hollow nanotubes, stimulated stem cell growth by phosphate ions supplied from BP oxidation, in situ immunoregulation, and osteogenesis induction by DEX molecules after implantation. Initial transplantation of the 3D spheroids in rat calvarial bone defect shows in vivo macrophage shifts to an M2 phenotype, leading to a pro‐healing microenvironment for regeneration. Prolonged implantation demonstrates outstanding in vivo neovascularization, osteointegration, and new bone regeneration. Therefore, these engineered 3D spheroids hold great promise for bone repair as they allow for stem cell delivery and provide immunoregulative and osteogenic signals within an all‐in‐one construct.

Intracellular pH regulation is vital for normal cellular function and is controlled by membrane H + and HCO 3 ‑ transporters. Most pH reporters have green fluorescence (i.e., BCECF, pHlorin) making simultaneous Ca 2+ , Cl ‐ , or other sensing (i.e., green fluorescent sensors) or GFP‐tags impossible. We previously reported a red‐fluorescent pH sensor ( pHire ) with an alkaline‐shifted pKa that increases fluorescence with increasing intracellular pH. pHire was transiently transfect into TM5 cells, but these fibroblast‐like cells did not address continuous expression in epithelial or excitable cells. Our objective was to evaluate pHire responses in multiple cell‐types and show that it can be used for fluorescent multiplexing. We selected pHire‐expressing rat pancreatic β‐cells (INS1 pHire ) and porcine kidney epithelial cells (LLCPK1 pHire ), and then FACS sorted to keep the top 10‐20% fluorescent cells. To test pHire’s usefulness for intact animals, we made transgenic, UAS‐pHire Drosophila , allowing pHire expression in specific tissues via genetic crossing with promoter‐GAL4 lines. By using perfusion chambers, we monitored cellular pHire‐responses to acid‐loading (NH 4 Cl pulse) and CO 2 /HCO 3 ‑ addition. Stable selection of pHire cell lines ensures single cells as well as contiguous cell layers for acid‐base transporter evaluation. Addition of NH 4 Cl quickly alkalinizes all pHire cells. Different acid‐recovery rates were found in individual cells with LLCPK1 pHire cells recovering faster than INS1 pHire cells. CO 2 /HCO 3 ‑ addition acidified both mammalian cell‐types. LLCPK1 pHire cells typically show a Na + dependent recovery (consistent with electrogenic Na + bicarbonate cotransporter, NBCe1, activity) that is absent from INS1 pHire cells. Infection of INS1 pHire cells with a NBCe1A‐GFP AAV allowed clear demonstration of NBCe1 activity. To test pHire’s stability in living animals, we next generated a UAS‐pHire transgenic Drosophila line, allowing pHire expression in specific tissues via genetic crossing with various promoter‐GAL4 lines. We tested renal epithelial (principal cells: Uro‐GAL4, CapaR‐GAL4; stellate cells: c724‐GAL4) and CNS (elav‐GAL4) expression. As observed in mammalian cells, NH 4 Cl acid‐loading showed robust and more uniform non‐HCO 3 ‑ recovery. The pH‐calibration curves for pHire in Drosophila cells were similar those obtained in mammalian cells (pKa>7.4). Thus, pHire characteristics are not changed by continuous expression in whole organisms. This study shows that the pHire sensor can be constitutively expressed into multiple cell lines or whole animals (flies) with an ideal pH range of pH 6 to 9. pHire may also be multiplexed with GFP labels and biosensors to allow real‐time, pH measurements with other ions of interest at a cellular scale. Finally, our data demonstrate that genetically encoded pHire is a reliable and useful tool for studying acid‐base transporters in a variety of cell‐types.