3D Stem Cell Spheroids with 2D Hetero-Nanostructures for In Vivo Osteogenic and Immunologic Modulated Bone Repair.

3D 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.

Bioactive Moldable Click Chemistry Polymer Cement with Nano-Hydroxyapatite and Growth Factor-Enhanced Posterolateral Spinal Fusion in a Rabbit Model.

Spinal injuries or diseases necessitate effective fusion solutions, and common clinical approaches involve autografts, allografts, and various bone matrix products, each with limitations. To address these challenges, we developed an innovative moldable click chemistry polymer cement that can be shaped by hand and self-cross-linked in situ for spinal fusion. This self-cross-linking cement, enabled by the bioorthogonal click reaction, excludes the need for toxic initiators or external energy sources. The bioactivity of the cement was promoted by incorporating nanohydroxyapatite and microspheres loaded with recombinant human bone morphogenetic protein-2 and vascular endothelial growth factor, fostering vascular induction and osteointegration. The release kinetics of growth factors, mechanical properties of the cement, and the ability of the scaffold to support in vitro cell proliferation and differentiation were evaluated. In a rabbit posterolateral spinal fusion model, the moldable cement exhibited remarkable induction of bone regeneration and effective bridging of spine vertebral bodies. This bioactive moldable click polymer cement therefore presents a promising biomaterial for spinal fusion augmentation, offering advantages in safety, ease of application, and enhanced bone regrowth.

Injectable bioactive poly(propylene fumarate) and polycaprolactone based click chemistry bone cement for spinal fusion in rabbits.

Degenerative 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.

Chronic intermittent ethanol exposure during adolescence produces sex- and age-dependent changes in anxiety and cognition without changes in microglia reactivity late in life.

Binge-like ethanol exposure during adolescence has been shown to produce long lasting effects in animal models including anxiety-like behavior that can last into young adulthood and impairments in cognition that can last throughout most of the lifespan. However, little research has investigated if binge-like ethanol exposure during adolescence produces persistent anxiety-like behavior and concomitantly impairs cognition late in life. Furthermore, few studies have investigated such behavioral effects in both female and male rats over the lifespan. Finally, it is yet to be determined if binge-like ethanol exposure during adolescence alters microglia activation in relevant brain regions late in life. In the present study female and male adolescent rats were exposed to either 3.0 or 5.0 g/kg ethanol, or water control, in a chronic intermittent pattern before being tested in the elevated plus maze and open field task over the next ∼18 months. Animals were then trained in a spatial reference task via the Morris water maze before having their behavioral flexibility tested. Finally, brains were removed, sectioned and presumptive microglia activation determined using autoradiography for [3H]PK11195 binding. Males, but not females, displayed an anxiety-like phenotype initially following the chronic intermittent ethanol exposure paradigm which resolved in adulthood. Further, males but not females had altered spatial reference learning and impaired behavioral flexibility late in life. Conversely, [3H]PK11195 binding was significantly elevated in females compared to males late in life and the level of microglia activation interacted as a function of sex and brain regions, but there was no long-term outcome related to adolescent alcohol exposure. These data further confirm that binge-like ethanol exposure during adolescence produces alterations in behavior that can last throughout the lifespan. In addition, the data suggest that microglia activation late in life is not exacerbated by prior binge-like ethanol exposure during adolescence but the expression is sex- and brain region-dependent across the lifespan.

Chronic Intermittent Ethanol Administration during Adolescence Produces Sex Dependent Impairments in Behavioral Flexibility and Survivability.

Chronic intermittent ethanol exposure during adolescence produces behavioral impairments and neurobiological changes that can last into young adulthood. One such behavioral impairment is reduced behavioral flexibility, a behavioral impairment that has been correlated with the risk for increased ethanol intake. In the current study, we investigated if chronic intermittent ethanol exposure during adolescence alters cognition, including behavioral flexibility, over a 22-month testing period. Female and male rats were treated with either 3.0 g/kg or 5.0 g/kg ethanol via gavage in a chronic intermittent fashion during adolescence and then tested every 4 to 5 months on a series of cognitive measures in the Morris water maze. Chronic intermittent ethanol selectively impaired behavioral flexibility in both female and male rats, although the pattern of results was different as a function of sex. In addition, female, but not male, rats were impaired in a short-term relearning test. Finally, male rats administered ethanol during adolescence were significantly more likely to not survive the 22-month experiment compared to female rats administered ethanol during adolescence. The current results demonstrate that adolescence is a unique period of development where chronic intermittent ethanol exposure produces long-lasting, selective cognitive impairments across the lifespan.

Chronic Intermittent Ethanol Exposure Alters Behavioral Flexibility in Aged Rats Compared to Adult Rats and Modifies Protein and Protein Pathways Related to Alzheimer's Disease.

Repeated excessive alcohol consumption increases the risk of developing cognitive decline and dementia. Hazardous drinking among older adults further increases such vulnerabilities. To investigate whether alcohol induces cognitive deficits in older adults, we performed a chronic intermittent ethanol exposure paradigm (ethanol or water gavage every other day 10 times) in 8-week-old young adult and 70-week-old aged rats. While spatial memory retrieval ascertained by probe trials in the Morris water maze was not significantly different between ethanol-treated and water-treated rats in both age groups after the fifth and tenth gavages, behavioral flexibility was impaired in ethanol-treated rats compared to water-treated rats in the aged group but not in the young adult group. We then examined ethanol-treatment-associated hippocampal proteomic and phosphoproteomic differences distinct in the aged rats. We identified several ethanol-treatment-related proteins, including the upregulations of the Prkcd protein level, several of its phosphosites, and its kinase activity and downregulation in the Camk2a protein level. Our bioinformatic analysis revealed notable changes in pathways involved in neurotransmission regulation, synaptic plasticity, neuronal apoptosis, and insulin receptor signaling. In conclusion, our behavioral and proteomic results identified several candidate proteins and pathways potentially associated with alcohol-induced cognitive decline in aged adults.

Acute ethanol administration produces larger spatial and nonspatial memory impairments in 29-33 month old rats compared to adult and 18-24 month old rats.

The average age of the population in many countries is continuing to increase. Older people continue to consume alcohol, often in a binge like fashion. Previous research has demonstrated that older human subjects and aged animal subjects have an increased sensitivity to the effects of ethanol on a variety of behaviors. However, it has yet to be determined if acute ethanol exposure impairs spatial and/or nonspatial memory to a greater extent in aged rats compared to adult rats. In the current studies we trained male rats ranging in age from young adult (2 months of age) to aged rats (29-33 months of age) in the standard nonspatial task followed by the standard spatial task in the Morris water maze. Only animals deemed "cognitively-spared", that is aged animals that learn as well as young animals, were administered one of two doses of moderate ethanol and had their memory tested 30 min later. Acute ethanol administration produced similar performance impairments in spatial and nonspatial memory in all cognitively-spared animals except for the 29-33 month old animals which showed a significantly greater cognitive impairment in both tasks. In addition, blood ethanol levels were similar across all ages. The present work adds to the growing literature on the selective effects of acute ethanol exposure in aged animals.