The Application of Ultrasmall Gold Nanoparticles (2 nm) Functionalized with Doxorubicin in Three-Dimensional Normal and Glioblastoma Organoid Models of the Blood-Brain Barrier.

Among brain tumors, glioblastoma (GBM) is very challenging to treat as chemotherapeutic drugs can only penetrate the brain to a limited extent due to the blood-brain barrier (BBB). Nanoparticles can be an attractive solution for the treatment of GBM as they can transport drugs across the BBB into the tumor. In this study, normal and GBM organoids comprising six brain cell types were developed and applied to study the uptake, BBB penetration, distribution, and efficacy of fluorescent, ultrasmall gold nanoparticles (AuTio-Dox-AF647s) conjugated with doxorubicin (Dox) and AlexaFluor-647-cadaverine (AF647) by confocal laser scanning microscopy (CLSM), using a mixture of dissolved doxorubicin and fluorescent AF647 molecules as a control. It was shown that the nanoparticles could easily penetrate the BBB and were found in normal and GBM organoids, while the dissolved Dox and AF647 molecules alone were unable to penetrate the BBB. Flow cytometry showed a reduction in glioblastoma cells after treatment with AuTio-Dox nanoparticles, as well as a higher uptake of these nanoparticles by GBM cells in the GBM model compared to astrocytes in the normal cell organoids. In summary, our results show that ultrasmall gold nanoparticles can serve as suitable carriers for the delivery of drugs into organoids to study BBB function.

Regenerative Medicine Therapies for Prevention of Abdominal Adhesions: A Scoping Review.

INTRODUCTION: Globally, abdominal adhesions constitute a significant burden of morbidity and mortality. They represent the commonest complication of abdominal operations with a lifelong risk of multiple pathologies, including adhesive small bowel obstruction, female infertility, and chronic pain. Adhesions represent a problem of the entire abdomen, forming at the time of injury and progressing through multiple complex pathways. Clinically available preventative strategies are limited to barrier technologies. Significant knowledge gaps persist in the characterization and mitigation of the involved molecular pathways underlying adhesion formation. Thus, the objectives of this scoping review are to describe the known molecular pathophysiology implicated in abdominal adhesion formation and summarize novel preclinical regenerative medicine preventative strategies for potential future clinical investigation. METHODS: A literature review was performed in accordance with the Preferred Reporting Items for Systematic Reviews Extension for Scoping Reviews. Included peer-reviewed publications were published within the last 5 y and contained in vivo preclinical experimental studies of postoperative adhesions with the assessment of underlying mechanisms of adhesion formation and successful therapy for adhesion prevention. Studies not involving regenerative medicine strategies were excluded. Data were qualitatively synthesized. RESULTS: A total of 1762 articles were identified. Of these, 1001 records were excluded by the described screening criteria. Sixty-eight full-text articles were evaluated for eligibility, and 11 studies were included for review. CONCLUSIONS: Novel and reliable preventative strategies are urgently needed. Recent experimental data propose novel regenerative medicine targets for adhesion prevention.

Extracellular vesicles from three dimensional culture of human placental mesenchymal stem cells ameliorated renal ischemia/reperfusion injury.

BACKGROUND: Three-dimensional (3D) culture has been reported to increase the therapeutic potential of mesenchymal stem cells (MSCs). The present study assessed the therapeutic efficacy of extracellular vesicles (EVs) from 3D cultures of human placental MSCs (hPMSCs) for acute kidney injury (AKI). METHODS: The supernatants from monolayer culture (2D) and 3D culture of hPMSCs were ultra-centrifuged for EVs isolation. C57BL/6 male mice were submitted to 45 min bilateral ischemia of kidney, followed by renal intra-capsular administration of EVs within a 72 h reperfusion period. Histological, immunohistochemical, and ELISA analyses of kidney samples were performed to evaluate cell death and inflammation. Kidney function was evaluated by measuring serum creatinine and urea nitrogen. The miRNA expression profiles of EVs from 2D and 3D culture of hPMSCs were evaluated using miRNA microarray analysis. RESULTS: The 3D culture of hPMSCs formed spheroids with different diameters depending on the cell density seeded. The hPMSCs produced significantly more EVs in 3D culture than in 2D culture. More importantly, injection of EVs from 3D culture of hPMSCs into mouse kidney with ischemia-reperfusion (I/R)-AKI was more beneficial in protecting from progression of I/R than those from 2D culture. The EVs from 3D culture of hPMSCs were more efficient against apoptosis and inflammation than those from 2D culture, which resulted in a reduction in tissue damage and amelioration of renal function. MicroRNA profiling analysis revealed that a set of microRNAs were significantly changed in EVs from 3D culture of hPMSCs, especially miR-93-5p. CONCLUSION: The EVs from 3D culture of hPMSCs have therapeutic potential for I/R-AKI.

Author Correction: Multicellular 3D Neurovascular Unit Model for Assessing Hypoxia and Neuroinflammation Induced Blood-Brain Barrier Dysfunction.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Transport of ultrasmall gold nanoparticles (2 nm) across the blood-brain barrier in a six-cell brain spheroid model.

The blood-brain barrier (BBB) is an efficient barrier for molecules and drugs. Multicellular 3D spheroids display reproducible BBB features and functions. The spheroids used here were composed of six brain cell types: Astrocytes, pericytes, endothelial cells, microglia cells, oligodendrocytes, and neurons. They form an in vitro BBB that regulates the transport of compounds into the spheroid. The penetration of fluorescent ultrasmall gold nanoparticles (core diameter 2 nm; hydrodynamic diameter 3-4 nm) across the BBB was studied as a function of time by confocal laser scanning microscopy, with the dissolved fluorescent dye (FAM-alkyne) as a control. The nanoparticles readily entered the interior of the spheroid, whereas the dissolved dye alone did not penetrate the BBB. We present a model that is based on a time-dependent opening of the BBB for nanoparticles, followed by a rapid diffusion into the center of the spheroid. After the spheroids underwent hypoxia (0.1% O2; 24 h), the BBB was more permeable, permitting the uptake of more nanoparticles and also of dissolved dye molecules. Together with our previous observations that such nanoparticles can easily enter cells and even the cell nucleus, these data provide evidence that ultrasmall nanoparticle can cross the blood brain barrier.

Multicellular 3D Neurovascular Unit Model for Assessing Hypoxia and Neuroinflammation Induced Blood-Brain Barrier Dysfunction.

The blood-brain barrier (BBB) is a dynamic component of the brain-vascular interface that maintains brain homeostasis and regulates solute permeability into brain tissue. The expression of tight junction proteins between adjacent endothelial cells and the presence of efflux proteins prevents entry of foreign substances into the brain parenchyma. BBB dysfunction, however, is evident in many neurological disorders including ischemic stroke, trauma, and chronic neurodegenerative diseases. Currently, major contributors to BBB dysfunction are not well understood. Here, we employed a multicellular 3D neurovascular unit organoid containing human brain microvascular endothelial cells, pericytes, astrocytes, microglia, oligodendrocytes and neurons to model the effects of hypoxia and neuroinflammation on BBB function. Organoids were cultured in hypoxic chamber with 0.1% O2 for 24 hours. Organoids cultured under this hypoxic condition showed increased permeability, pro-inflammatory cytokine production, and increased oxidative stress. The anti-inflammatory agents, secoisolariciresinol diglucoside and 2-arachidonoyl glycerol, demonstrated protection by reducing inflammatory cytokine levels in the organoids under hypoxic conditions. Through the assessment of a free radical scavenger and an anti-inflammatory endocannabinoid, we hereby report the utility of the model in drug development for drug candidates that may reduce the effects of ROS and inflammation under disease conditions. This 3D organoid model recapitulates characteristics of BBB dysfunction under hypoxic physiological conditions and when exposed to exogenous neuroinflammatory mediators and hence may have potential in disease modeling and therapeutic development.

Implementation Guide for Rapid Integration of an Outpatient Telemedicine Program During the COVID-19 Pandemic.

BACKGROUND: In the novel coronavirus disease 2019 (COVID-19) pandemic, social distancing has been necessary to help prevent disease transmission. As a result, medical practices have limited access to in-person visits. This poses a challenge to maintain appropriate patient care while preventing a substantial backlog of patients once stay-at-home restrictions are lifted. In practices that are naïve to telehealth as an alternative option, providers and staff are experiencing challenges with telemedicine implementation. We aim to provide a comprehensive guide on how to rapidly integrate telemedicine into practice during a pandemic. METHODS: We built a toolkit that details the following 8 essential components to successful implementation of a telemedicine platform: provider and staff training, patient education, an existing electronic medical record system, patient and provider investment in hardware, billing and coding integration, information technology support, audiovisual platforms, and patient and caregiver participation. RESULTS: Rapid integration of telemedicine in our practice was required to be compliant with our institution's COVID-19 task force. Within 3 days of this declaration, our large specialty-care clinic converted to a telemedicine platform and we completed 638 visits within the first month of implementation. CONCLUSIONS: Effective and efficient integration of a telemedicine program requires extensive staff and patient education, accessory platforms to facilitate video and audio communication, and adoption of new billing codes that are outlined in this toolkit.

Ultrasmall gold nanoparticles (2 nm) can penetrate and enter cell nuclei in an in vitro 3D brain spheroid model.

The neurovascular unit (NVU) is a complex functional and anatomical structure composed of endothelial cells and their blood-brain barrier (BBB) forming tight junctions. It represents an efficient barrier for molecules and drugs. However, it also prevents a targeted transport for the treatment of cerebral diseases. The uptake of ultrasmall nanoparticles as potential drug delivery agents was studied in a three-dimensional co-culture cell model (3D spheroid) composed of primary human cells (astrocytes, pericytes, endothelial cells). Multicellular 3D spheroids show reproducible NVU features and functions. The spheroid core is composed mainly of astrocytes, covered with pericytes, while brain endothelial cells form the surface layer, establishing the NVU that regulates the transport of molecules. After 120 h cultivation, the cells self-assemble into a 350 µm spheroid as shown by confocal laser scanning microscopy. The passage of different types of fluorescent ultrasmall gold nanoparticles (core diameter 2 nm) both into the spheroid and into three constituting cell types was studied by confocal laser scanning microscopy. Three kinds of covalently fluorophore-conjugated gold nanoparticles were used: One with fluorescein (FAM), one with Cy3, and one with the peptide CGGpTPAAK-5,6-FAM-NH2. In 2D cell co-culture experiments, it was found that all three kinds of nanoparticles readily entered all three cell types. FAM- and Cy3-labelled nanoparticles were able to enter the cell nucleus as well. The three dissolved dyes alone were not taken up by any cell type. A similar situation evolved with 3D spheroids: The three kinds of nanoparticles entered the spheroid, but the dissolved dyes did not. The presence of a functional blood-brain barrier was demonstrated by adding histamine to the spheroids. In that case, the blood-brain barrier opened, and dissolved dyes like a FITC-labelled antibody and FITC alone entered the spheroid. In summary, our results qualify ultrasmall gold nanoparticles as suitable carriers for imaging or drug delivery into brain cells (sometimes including the nucleus), brain cell spheroids, and probably also into the brain. STATEMENT OF SIGNIFICANCE: 3D brain spheroid model and its permeability by ultrasmall gold nanoparticles. We demonstrate that ultrasmall gold nanoparticles can easily penetrate the constituting cells and sometimes even enter the cell nucleus. They can also enter the interior of the blood-brain barrier model. In contrast, small molecules like fluorescing dyes are not able to do that. Thus, ultrasmall gold nanoparticles can serve as carriers of drugs or for imaging inside the brain.

A Case Report of an Obstructing Ureteral Nephrogenic Adenoma in a Child Managed With Open Ileal Ureter.

Nephrogenic adenoma is a rare, benign lesion that can be encountered anywhere along the urinary tract. It is associated with genitourinary trauma, chronic inflammation, genitourinary surgery, renal transplant, urolithiasis, and radiation. In children, these lesions are almost exclusively found in the bladder. However, we report an unusual case of a 15-year-old boy with no prior urologic history who presented with an obstructing right ureteral nephrogenic adenoma that required an ileal ureter interposition and right ureterectomy.

Therapeutic Mesenchymal Stromal Cells for Immunotherapy and for Gene and Drug Delivery.

Mesenchymal stromal cells (MSCs) possess several fairly unique properties that, when combined, make them ideally suited for cellular-based immunotherapy and as vehicles for gene and drug delivery for a wide range of diseases and disorders. Key among these are: (1) their relative ease of isolation from a variety of tissues; (2) the ability to be expanded in culture without a loss of functionality, a property that varies to some degree with tissue source; (3) they are relatively immune-inert, perhaps obviating the need for precise donor/recipient matching; (4) they possess potent immunomodulatory functions that can be tailored by so-called licensing in vitro and in vivo; (5) the efficiency with which they can be modified with viral-based vectors; and (6) their almost uncanny ability to selectively home to damaged tissues, tumors, and metastases following systemic administration. In this review, we summarize the latest research in the immunological properties of MSCs, their use as immunomodulatory/anti-inflammatory agents, methods for licensing MSCs to customize their immunological profile, and their use as vehicles for transferring both therapeutic genes in genetic disease and drugs and genes designed to destroy tumor cells.

The hematopoietic system in the context of regenerative medicine.

Hematopoietic stem cells (HSC) represent the prototype stem cell within the body. Since their discovery, HSC have been the focus of intensive research, and have proven invaluable clinically to restore hematopoiesis following inadvertent radiation exposure and following radio/chemotherapy to eliminate hematologic tumors. While they were originally discovered in the bone marrow, HSC can also be isolated from umbilical cord blood and can be "mobilized" peripheral blood, making them readily available in relatively large quantities. While their ability to repopulate the entire hematopoietic system would already guarantee HSC a valuable place in regenerative medicine, the finding that hematopoietic chimerism can induce immunological tolerance to solid organs and correct autoimmune diseases has dramatically broadened their clinical utility. The demonstration that these cells, through a variety of mechanisms, can also promote repair/regeneration of non-hematopoietic tissues as diverse as liver, heart, and brain has further increased their clinical value. The goal of this review is to provide the reader with a brief glimpse into the remarkable potential HSC possess, and to highlight their tremendous value as therapeutics in regenerative medicine.

Applicability and safety of in vitro skin expansion using a skin bioreactor: a clinical trial.

BACKGROUND: Tissue expansion is an effective and valuable technique for the reconstruction of large skin lesions and scars. This study aimed to evaluate the applicability and safety of a newly designed skin expanding bioreactor system for maximizing the graft area and minimizing the donor site area. METHODS: A computer-controlled biaxial skin bioreactor system was used to expand skin in two directions while the culture media was changed daily. The aim was to achieve an expansion speed that enabled the skin to reach twice its original area in two weeks or less. Skin expansion and subsequent grafting were performed for 10 patients, and each patient was followed for 6 months postoperatively for clinical evaluation. Scar evaluation was performed through visual assessment and by using photos. RESULTS: The average skin expansion rate was 10.54%±6.25%; take rate, 88.89%±11.39%; and contraction rate, 4.2%±2.28% after 6 months. Evaluation of the donor and recipient sites by medical specialists resulted in an average score of 3.5 (out of a potential maximum of 5) at 3 months, and 3.9 at 6 months. The average score for patient satisfaction of the donor site was 6.2 (out of a potential maximum of 10), and an average score of 5.2 was noted for the recipient site. Histological examination performed before and after the skin expansion revealed an increase in porosity of the dermal layer. CONCLUSIONS: This study confirmed the safety and applicability of the in vitro skin bioreactor, and further studies are needed to develop methods for increasing the skin expansion rate.

Segmental ulcerated perineal hemangioma of infancy: a complex case of PELVIS syndrome successfully treated using a multidisciplinary approach.

We report a case of PELVIS (perineal hemangioma, external genital malformations, lipomyelomeningocele, vesicorenal abnormalities, imperforate anus and skin tag) syndrome in which hemangioma in the perineal area was misdiagnosed at birth as diaper rash. Investigations revealed associated vesicorenal and spinal abnormalities. We emphasize careful diagnosis of suspicious lesions at birth and confirm the successful use of propranolol in treating ulcerated segmental hemangiomas.

Human mid-trimester amniotic fluid stem cells cultured under embryonic stem cell conditions with valproic acid acquire pluripotent characteristics.

Human mid-trimester amniotic fluid stem cells (AFSC) have promising applications in regenerative medicine, being broadly multipotent with an intermediate phenotype between embryonic (ES) and mesenchymal stem cells (MSC). Despite this propluripotent phenotype, AFSC are usually cultured in adherence in a serum-based expansion medium, and how expansion in conditions sustaining pluripotency might affect their phenotype remains unknown. We recently showed that early AFSC from first trimester amniotic fluid, which endogenously express Sox2 and Klf4, can be reprogrammed to pluripotency without viral vectors using the histone deacetylase inhibitor valproic acid (VPA). Here, we show that mid-trimester AFSC cultured under MSC conditions contained a subset of cells endogenously expressing telomerase, CD24, OCT4, C-MYC, and SSEA4, but low/null levels of SOX2, NANOG, KLF4, SSEA3, TRA-1-60, and TRA-1-81, with cells unable to form embryoid bodies (EBs) or teratomas. In contrast, AFSC cultured under human ESC conditions were smaller in size, grew faster, formed colonies, upregulated OCT4 and C-MYC, and expressed KLF4 and SOX2, but not NANOG, SSEA3, TRA-1-60, and TRA-1-81. Supplementation with VPA for 5 days further upregulated OCT4, KLF4, and SOX2, and induced expression of NANOG, SSEA3, TRA-1-60, and TRA-1-81, with cells now able to form EBs and teratomas. We conclude that human mid-trimester AFSC, which may be isolated autologously during pregnancy without ethics restriction, can acquire pluripotent characteristics without the use of ectopic factors. Our data suggest that this medium-dependant approach to pluripotent mid-trimester AFSC reflects true reprogramming and not the selection of prepluripotent cells.