Development of a Single-Neurosphere Culture to Assess Radiation Toxicity and Pre-Clinical Cancer Combination Therapy Safety.

Radiation therapy (RT) is a crucial treatment modality for central nervous system (CNS) tumors but toxicity to healthy CNS tissues remains a challenge. Additionally, environmental exposure to radiation during nuclear catastrophes or space travel presents a risk of CNS toxicity. However, the underlying mechanisms of radiation-induced CNS toxicity are not fully understood. Neural progenitor cells (NPCs) are highly radiosensitive, resulting in decreased neurogenesis in the hippocampus. This study aimed to characterize a novel platform utilizing rat NPCs cultured as 3D neurospheres (NSps) to screen the safety and efficacy of experimental drugs with and without radiation exposure. The effect of radiation on NSp growth and differentiation was assessed by measuring sphere volume and the expression of neuronal differentiation markers Nestin and GFAP and proliferation marker Ki67. Radiation exposure inhibited NSp growth, decreased proliferation, and increased GFAP expression, indicating astrocytic differentiation. RNA sequencing analysis supported these findings, showing upregulation of Notch, BMP2/4, S100b, and GFAP gene expression during astrogenesis. By recapitulating radiation-induced toxicity and astrocytic differentiation, this single-NSp culture system provides a high-throughput preclinical model for assessing the effects of various radiation modalities and evaluates the safety and efficacy of potential therapeutic interventions in combination with radiation.

Premature Neural Progenitor Cell Differentiation Into Astrocytes in Retinoic Acid-Induced Spina Bifida Rat Model.

During embryonic spinal cord development, neural progenitor cells (NPCs) generate three major cell lines: neurons, oligodendrocytes, and astrocytes at precise times and locations within the spinal cord. Recent studies demonstrate early astrogenesis in animal models of spina bifida, which may play a role in neuronal dysfunction associated with this condition. However, to date, the pathophysiological mechanisms related to this early astrocytic response in spina bifida are poorly understood. This study aimed to characterize the development of early astrogliosis over time from Pax6+, Olig2+, or Nkx2.2+ NPCs using a retinoic acid-induced spina bifida rat model. At three gestational ages (E15, E17, and E20), spinal cords from fetuses with retinoic acid-induced spina bifida, their healthy sibling controls, or fetuses treated with the vehicle control were analyzed. Results indicated that premature astrogliosis and astrocytic activation were associated with an altered presence of Pax6+, Olig2+, and Nkx2.2+ NPCs in the lesion compared to the controls. Finally, this response correlated with an elevation in genes involved in the Notch-BMP signaling pathway. Taken together, changes in NPC patterning factor expression with Notch-BMP signaling upregulation may be responsible for the altered astrogenesis patterns observed in the spinal cord in a retinoic acid-induced spina bifida model.

Dural substitutes for spina bifida repair: past, present, and future.

PURPOSE: The use of materials to facilitate dural closure during spina bifida (SB) repair has been a highly studied aspect of the surgical procedure. The overall objective of this review is to present key findings pertaining to the success of the materials used in clinical and pre-clinical studies. Additionally, this review aims to aid fetal surgeons as they prepare for open or fetoscopic prenatal SB repairs. METHODS: Relevant publications centered on dural substitutes used during SB repair were identified. Important information from each article was extracted including year of publication, material class and sub-class, animal model used in pre-clinical studies, whether the repair was conducted pre-or postnatally, the bioactive agent delivered, and key findings from the study. RESULTS: Out of 1,121 publications, 71 were selected for full review. We identified the investigation of 33 different patches where 20 and 63 publications studied synthetic and natural materials, respectively. From this library, 43.6% focused on clinical results, 36.6% focused on pre-clinical results, and 19.8% focused on tissue engineering approaches. Overall, the use of patches, irrespective of material, have shown to successfully protect the spinal cord and most have shown promising survival and neurological outcomes. CONCLUSION: While most have shown significant promise as a therapeutic strategy in both clinical and pre-clinical studies, none of the patches developed so far are deemed perfect for SB repair. Therefore, there is an opportunity to develop new materials and strategies that aim to overcome these challenges and further improve the outcomes of SB patients.

Time Course Transcriptome Analysis of Spina Bifida Progression in Fetal Rats.

A better understanding of the transcriptomic modifications that occur in spina bifida may lead to identify mechanisms involved in the progression of spina bifida in utero and the development of new therapeutic strategies that aid in spinal cord regeneration after surgical interventions. In this study, RNA-sequencing was used to identify differentially expressed genes in fetal spinal cords from rats with retinoic acid-induced spina bifida at E15, E17, and E20. Gene ontology, KEGG, and protein-protein interaction analysis were conducted to predict pathways involved in the evolution of the disease. Approximately 3000, 1000 and 300 genes were differentially expressed compared to the control groups at E15, E17 and E20, respectively. Overall, the results suggest common alterations in certain pathways between gestational time points, such as upregulation in p53 and sonic hedgehog signaling at E15 and E17 and downregulation in the myelin sheath at E17 and E20. However, there were other modifications specific to gestational time points, including skeletal muscle development at E15, downregulated glucose metabolism at E17, and upregulated inflammation at E20. In conclusion, this work provides evidence that gestational age during spina bifida repair may be a significant variable to consider during the development of new regenerative therapeutics approaches.

Modulation of adipocyte size and fat pad weight via resveratrol releasing scaffolds implanted into the epididymal adipose tissue.

Lipid overload of the adipose tissue, which can be caused by overnutrition, underlies metabolic disease. We hypothesized that increasing the energy demand of adipose tissue is a promising strategy to combat excessive lipid accumulation. Resveratrol, a natural polyphenol, activates lipid catabolism in fat tissue; however, its clinical success is hindered by poor bioavailability. Here, we implanted resveratrol releasing poly(lactide-co-glycolide) scaffolds into epididymal fat to overcome its poor bioavailability with the goal of enhancing local lipid catabolism. In lean mice, resveratrol scaffolds decreased adipocyte size relative to scaffolds with no drug, a response that correlated with AMP kinase activation. Immunohistochemistry indicated that macrophages and multinucleated giant cells within the scaffold expressed carnitine palmitoyltransferase 1 (CPT1) at higher levels than other cells in the adipose tissue. Furthermore, resveratrol increased CPT1 levels in cultured macrophages. Taken together, we propose that resveratrol scaffolds decrease adipocyte size because resveratrol increases lipid utilization in scaffold-infiltrating immune cells, possibly through elevating CPT1 levels or activity. In a follow-up study, mice that received resveratrol scaffolds 28-day prior to a high-fat diet exhibited decreased weight gain, adipose tissue expansion, and adipocyte hypertrophy compared to mice with control scaffolds. Notably, this scaffold-based strategy required a single resveratrol administration compared to the daily regiment generally needed for oral administration. These results indicate that localized delivery of metabolism modulating agents to the adipose tissue may overcome issues with bioavailability and that the role of biomaterials should be further investigated in this therapeutic strategy for metabolic disease.

Scaffold Implant Into the Epididymal Adipose Tissue Protects Mice From High Fat Diet Induced Ectopic Lipid Accumulation and Hyperinsulinemia.

Ectopic lipid accumulation, the deposition of lipids in lean tissue, is linked to type 2 diabetes through an association with insulin resistance. It occurs when adipose tissue fails to meet lipid storage needs and there is lipid spillover into tissues not equipped to store them. Ectopic lipid contributes to organ dysfunction because lipids can interfere with insulin signaling and other signaling pathways. Clinical studies indicate that decreasing ectopic lipids through diet and exercise is effective in treating type 2 diabetes; however, its prevalence continues to rise. We propose that strategies to improve lipid handling in the adipose tissue would be adjunctive to healthy lifestyle modification and may address difficulties in treating type 2 diabetes and other syndromes spurred by ectopic lipid. Herein, we investigate biomaterial implants as a means to increase lipid utilization in adipose tissue through the recruitment of highly metabolic cells. Poly(lactide-co-glycolide) scaffolds were implanted into the epididymal fat of mice fed a high fat diet that overwhelms the adipose tissue and promotes ectopic lipid accumulation. Over 5 weeks, mice with scaffolds gained less weight compared to mice without scaffolds and were protected from hyperinsulinemia. These effects correlated with a 53% decrease in triglyceride in the gastrocnemius and a 25% decrease in the liver. Scaffolds increased CPT1A protein levels in the epididymal fat and histology revealed high expression of CTP1A in the cells infiltrating the scaffold relative to the rest of the fat pad. In addition, lacing the scaffold with resveratrol increased CPT1A expression in the epididymal fat over scaffolds with no drug; however, this did not result in further decreases in weight gain or ectopic lipid. Mechanistically, we propose that the cellular activity caused by scaffold implant mitigates the lipid load imposed by the high fat diet and leads to a substantial decrease in lipid accumulation in the muscle and liver. In conclusion, this study establishes that a tissue engineering approach to modulate lipid utilization in the epididymal fat tissue can mitigate ectopic lipid accumulation in mice fed a high fat diet with positive effects on weight gain and whole-body insulin resistance.

Development of microparticles for controlled release of resveratrol to adipose tissue and the impact of drug loading on particle morphology and drug release.

Resveratrol is a small molecule produced by various plants with a remarkable range of beneficial functions in animals. One of these is stimulating signaling pathways in adipose tissue that protect against obesity. Unfortunately, resveratrol suffers from poor bioavailability that inhibits its accumulation in target tissues, including fat, thus hindering the realization of its therapeutic potential. To address this, we are developing biodegradable microparticles as drug depots for controlled release of resveratrol within fat. In this study, resveratrol was encapsulated into poly(lactide-co-glycolide) microparticles using an oil-in-water emulsion/solvent evaporation technique. The oil phase consisted of resveratrol and poly(lactide-co-glycolide) dissolved in a mixture of dichloromethane and ethanol; meanwhile, the aqueous phase contained poly(vinyl alcohol) as the emulsifier. Increasing ethanol's volume ratio increased resveratrol's solubility in the oil phase and particle drug loading. The maximal loading achieved was 65 µg/mg (6.5%) and occurred when the ethanol to dichloromethane ratio was 1:3. Under these conditions, particles exhibited ruffled surfaces, which resulted in variable drug release over the first three days of a six-week release assay. By decreasing resveratrol and ethanol in the oil phase and increasing poly(vinyl alcohol) in the aqueous phase, smooth particles were achieved, but they suffered a 15-25-fold decrease in drug loading depending on size. Small particles exhibited higher drug loading and burst drug release compared to larger particles because of their higher specific surface area. Utilizing mild chemistry, we functionalized poly(vinyl alcohol) with fluorescein isothiocyanate and demonstrated that encapsulation of resveratrol in the particle decreases the amount of fluorescent polymer on the particle surface, suggesting resveratrol displaces the emulsifier during particle formation. Taken together, resveratrol can be encapsulated into poly(lactide-co-glycolide) microparticles, but it accumulates at the particle surface impacting drug loading, surface roughness, and drug release.

The host response to poly(lactide-co-glycolide) scaffolds protects mice from diet induced obesity and glucose intolerance.

Underlying metabolic disease is poor adipose tissue function characterized by impaired glucose tolerance and low expression of health promoting adipokines. Currently, no treatments specifically target the adipose tissue and we are investigating polymer scaffolds for localized drug delivery as a therapeutic platform. In this work we implanted porous poly(lactide-co-glycolide) scaffolds into the epididymal fat of mice. Surprisingly, "empty" scaffolds decreased blood glucose levels in healthy mice as well as epididymal fat pad size. By injecting a fluorescent glucose tracer into mice, we determined that glucose uptake increases by 60% in epididymal fat pads with scaffolds; in contrast, glucose uptake was not elevated in other major metabolic organs, suggesting the enhanced glucose uptake at the scaffold implant site was responsible for decreased blood glucose levels. Histology indicated increased cellularity and tissue remodeling around the scaffold and we found increased expression of glucose transporter 1 and insulin-like growth factor 1, which are proteins involved in wound healing that can also modulate blood glucose levels through their promotion of glucose uptake. Regarding clinical translation, "empty" scaffolds decreased obesity and improved glucose tolerance in mice fed a high fat diet. These findings demonstrate increased cellular activity in the adipose tissue, such as that associated with the host response to biomaterial implant, is beneficial in mice suffering from metabolic complications of over nutrition, possibly because it mitigates the positive energy balance that leads to the obese, diabetic state. More broadly, this work reaffirms that in addition to the local host response typically investigated, biomaterial implant has systemic physiological effects and suggests that there may be implications for therapy.

Resveratrol Delivery from Porous Poly(lactide- co-glycolide) Scaffolds Promotes an Anti-Inflammatory Environment within Visceral Adipose Tissue.

As biomaterial therapies emerge to address adipose tissue dysfunction that underlies metabolic disease, the immune response to these systems must be established. As a potential therapy, we are investigating resveratrol delivery from porous poly(lactide- co-glycolide) scaffolds designed to integrate with adipose tissue. Resveratrol was selected for its ability to protect mice and primates from high fat diet and broad anti-inflammatory properties. Herein, we report fabrication of scaffolds with high resveratrol loading that are stable and active for up to one year. In vitro release profiles indicate that drug release is biphasic with a burst release over 3 days followed by a plateau. Surprisingly, we find that PLG scaffolds implanted into adipose tissue of mice promote an anti-inflammatory environment characterized by high arginase-1 and low TNF-α and IL-6 compared to naïve unmanipulated fat. Resveratrol delivery from the scaffold augments this anti-inflammatory environment by decreasing monocyte and lymphocyte numbers at the implant site and increasing expression of IL-10 and IL-13, cytokines that promote healthy adipose tissue. In terms of therapeutic applications, implant of scaffolds designed to release resveratrol into the visceral fat decreases MCP-1 expression in mice fed a high fat diet, a molecule that drives both local and systemic inflammation during obesity. Taken together, resveratrol delivery to adipose tissue using poly(lactide- co-glycolide) scaffolds is a promising therapeutic strategy for the treatment of adipose tissue inflammation that drives metabolic disease.