Engineered extracellular vesicle-based gene therapy for the treatment of discogenic back pain.

Painful musculoskeletal disorders such as intervertebral disc (IVD) degeneration associated with chronic low back pain (termed "Discogenic back pain", DBP), are a significant socio-economic burden worldwide and contribute to the growing opioid crisis. Yet there are very few if any successful interventions that can restore the tissue's structure and function while also addressing the symptomatic pain. Here we have developed a novel non-viral gene therapy, using engineered extracellular vesicles (eEVs) to deliver the developmental transcription factor FOXF1 to the degenerated IVD in an in vivo model. Injured IVDs treated with eEVs loaded with FOXF1 demonstrated robust sex-specific reductions in pain behaviors compared to control groups. Furthermore, significant restoration of IVD structure and function in animals treated with FOXF1 eEVs were observed, with significant increases in disc height, tissue hydration, proteoglycan content, and mechanical properties. This is the first study to successfully restore tissue function while modulating pain behaviors in an animal model of DBP using eEV-based non-viral delivery of transcription factor genes. Such a strategy can be readily translated to other painful musculoskeletal disorders.

Nonviral overexpression of Scleraxis or Mohawk drives reprogramming of degenerate human annulus fibrosus cells from a diseased to a healthy phenotype.

Background: Intervertebral disc (IVD) degeneration is a major contributor to low back pain (LBP), yet there are no clinical therapies targeting the underlying pathology. The annulus fibrosus (AF) plays a critical role in maintaining IVD structure/function and undergoes degenerative changes such as matrix catabolism and inflammation. Thus, therapies targeting the AF are crucial to fully restore IVD function. Previously, we have shown nonviral delivery of transcription factors to push diseased nucleus pulposus cells to a healthy phenotype. As a next step in a proof-of-concept study, we report the use of Scleraxis (SCX) and Mohawk (MKX), which are critical for the development, maintenance, and regeneration of the AF and may have therapeutic potential to induce a healthy, pro-anabolic phenotype in diseased AF cells. Methods: MKX and SCX plasmids were delivered via electroporation into diseased human AF cells from autopsy specimens and patients undergoing surgery for LBP. Transfected cells were cultured over 14 days and assessed for cell morphology, viability, density, gene expression of key phenotypic, inflammatory, matrix, pain markers, and collagen accumulation. Results: AF cells demonstrated a fibroblastic phenotype posttreatment. Moreover, transfection of SCX and MKX resulted in significant upregulation of the respective genes, as well as SOX9. Transfected autopsy cells demonstrated upregulation of core extracellular matrix markers; however, this was observed to a lesser effect in surgical cells. Matrix-degrading enzymes and inflammatory cytokines were downregulated, suggesting a push toward a pro-anabolic, anti-inflammatory phenotype. Similarly, pain markers were downregulated over time in autopsy cells. At the protein level, collagen content was increased in both MKX and SCX transfected cells compared to controls. Conclusions: This exploratory study demonstrates the potential of MKX or SCX to drive reprogramming in mild to moderately degenerate AF cells from autopsy and severely degenerate AF cells from surgical patients toward a healthy phenotype and may be a potential nonviral gene therapy for LBP.

Engineered Extracellular Vesicle-Based Therapies for Valvular Heart Disease.

Introduction: Valvular heart disease represents a significant burden to the healthcare system, with approximately 5 million cases diagnosed annually in the US. Among these cases, calcific aortic stenosis (CAS) stands out as the most prevalent form of valvular heart disease in the aging population.  CAS is characterized by the progressive calcification of the aortic valve leaflets, leading to valve stiffening. While aortic valve replacement is the standard of care for CAS patients, the long-term durability of prosthetic devices is poor, calling for innovative strategies to halt  or reverse disease progression. Here, we explor the potential use of novel extracellular vesicle (EV)-based nanocarriers for delivering molecular payloads to the affected valve tissue. This approach aims to reduce inflammation and potentially promote resorption of the calcified tissue. Methods: Engineered EVs loaded with the reprogramming myeloid transcription factors, CEBPA and Spi1, known to mediate the transdifferentiation of committed endothelial cells into macrophages. We evaluated the ability of these engineered EVs to deliver DNA and transcripts encoding CEBPA and Spil into calcified aortic valve tissue obtained from patients undergoing valve replacement due to aortic stenosis. We also investigated whether these EVs could induce the transdifferentiation of endothelial cells into macrophage-like cells. Results: Engineered EVs loaded with CEBPA + Spi1 were successfully derived from human dermal fibroblasts. Peak EV loading was found to be at 4 h after nanotransfection of donor cells.  These CEBPA + Spi1 loaded EVs effectively transfected aortic valve cells, resulting in the successful induction of transdifferentiation, both in vitro with  endothelial cells and ex vivo with valvular endothelial cells, leading to the development of anti-inflammatory macrophage-like cells. Conclusions: Our findings highlight the potential of engineered EVs as a next generation nanocarrier to target aberrant calcifications on diseased heart valves. This development holds promise as a novel therapy for high-risk patients who may not be suitable candidates for valve replacement surgery. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00783-x.

ICAM-1-decorated extracellular vesicles loaded with miR-146a and Glut1 drive immunomodulation and hinder tumor progression in a murine model of breast cancer.

Tumor-associated immune cells play a crucial role in cancer progression. Myeloid-derived suppressor cells (MDSCs), for example, are immature innate immune cells that infiltrate the tumor to exert immunosuppressive activity and protect cancer cells from the host's immune system and/or cancer-specific immunotherapies. While tumor-associated immune cells have emerged as a promising therapeutic target, efforts to counter immunosuppression within the tumor niche have been hampered by the lack of approaches that selectively target the immune cell compartment of the tumor, to effectively eliminate "tumor-protecting" immune cells and/or drive an "anti-tumor" phenotype. Here we report on a novel nanotechnology-based approach to target tumor-associated immune cells and promote "anti-tumor" responses in a murine model of breast cancer. Engineered extracellular vesicles (EVs) decorated with ICAM-1 ligands and loaded with miR-146a and Glut1, were biosynthesized (in vitro or in vivo) and administered to tumor-bearing mice once a week for up to 5 weeks. The impact of this treatment modality on the immune cell compartment and tumor progression was evaluated via RT-qPCR, flow cytometry, and histology. Our results indicate that weekly administration of the engineered EVs (i.e., ICAM-1-decorated and loaded with miR-146a and Glut1) hampered tumor progression compared to ICAM-1-decorated EVs with no cargo. Flow cytometry analyses of the tumors indicated a shift in the phenotype of the immune cell population toward a more pro-inflammatory state, which appeared to have facilitated the infiltration of tumor-targeting T cells, and was associated with a reduction in tumor size and decreased metastatic burden. Altogether, our results indicate that ICAM-1-decorated EVs could be a powerful platform nanotechnology for the deployment of immune cell-targeting therapies to solid tumors.

Engineered Vasculogenic Extracellular Vesicles Drive Nonviral Direct Conversions of Human Dermal Fibroblasts into Induced Endothelial Cells and Improve Wound Closure.

Vasculogenic cell therapies have emerged as a powerful tool to increase vascularization and promote tissue repair/regeneration. Current approaches to cell therapies, however, rely mostly on progenitor cells, which pose significant risks (e.g., uncontrolled differentiation, tumorigenesis, and genetic/epigenetic abnormalities). Moreover, reprogramming methodologies used to generate induced endothelial cells (iECs) from induced pluripotent stem cells rely heavily on viral vectors, which pose additional translational limitations. This work describes the development of engineered human extracellular vesicles (EVs) capable of driving reprogramming-based vasculogenic therapies without the need for progenitor cells and/or viral vectors. The EVs were derived from primary human dermal fibroblasts (HDFs), and were engineered to pack transcription factor genes/transcripts of ETV2, FLI1, and FOXC2 (EFF). Our results indicate that in addition of EFF, the engineered EVs were also loaded with transcripts of angiogenic factors (e.g., VEGF-A, VEGF-KDR, FGF2). In vitro and in vivo studies indicate that such EVs effectively transfected HDFs and drove direct conversions towards iECs within 7-14 days. Finally, wound healing studies in mice indicate that engineered EVs lead to improved wound closure and vascularity. Altogether, our results show the potential of engineered human vasculogenic EVs to drive direct reprogramming processes of somatic cells towards iECs, and facilitate tissue repair/regeneration.

In Situ Deployment of Engineered Extracellular Vesicles into the Tumor Niche via Myeloid-Derived Suppressor Cells.

Extracellular vesicles (EVs) have emerged as a promising carrier system for the delivery of therapeutic payloads in multiple disease models, including cancer. However, effective targeting of EVs to cancerous tissue remains a challenge. Here, it is shown that nonviral transfection of myeloid-derived suppressor cells (MDSCs) can be leveraged to drive targeted release of engineered EVs that can modulate transfer and overexpression of therapeutic anticancer genes in tumor cells and tissue. MDSCs are immature immune cells that exhibit enhanced tropism toward tumor tissue and play a role in modulating tumor progression. Current MDSC research has been mostly focused on mitigating immunosuppression in the tumor niche; however, the tumor homing abilities of these cells present untapped potential to deliver EV therapeutics directly to cancerous tissue. In vivo and ex vivo studies with murine models of breast cancer show that nonviral transfection of MDSCs does not hinder their ability to home to cancerous tissue. Moreover, transfected MDSCs can release engineered EVs and mediate antitumoral responses via paracrine signaling, including decreased invasion/metastatic activity and increased apoptosis/necrosis. Altogether, these findings indicate that MDSCs can be a powerful tool for the deployment of EV-based therapeutics to tumor tissue.

Delayed Processing of Secretin-Induced Pancreas Fluid Influences the Quality and Integrity of Proteins and Nucleic Acids.

OBJECTIVES: Endoscopic pancreatic function tests are used to diagnose pancreatic diseases and are a viable source for the discovery of biomarkers to better characterize pancreatic disorders. However, pancreatic fluid (PF) contains active enzymes that degrade biomolecules. Therefore, we tested how preservation methods and time to storage influence the integrity and quality of proteins and nucleic acids. METHODS: We obtained PF from 9 subjects who underwent an endoscopic pancreatic function test. Samples were snap frozen at the time of collection; after 1, 2, and 4 hours on ice; or after storage overnight at 4°C with or without RNase or protease inhibitors (PIs). Electrophoresis and mass spectrometry analysis determined protein abundance and quality, whereas nucleic acid integrity values determined DNA and RNA degradation. RESULTS: Protein degradation increased after 4 hours on ice and DNA degradation after 2 hours on ice. Adding PIs delayed degradation. RNA was significantly degraded under all conditions compared with the snap frozen samples. Isolated RNA from PF-derived exosomes exhibited similar poor quality as RNA isolated from matched PF samples. CONCLUSIONS: Adding PIs immediately after collecting PF and processing the fluid within 4 hours of collection maintains the protein and nucleic acid integrity for use in downstream molecular analyses.

Guided migration analyses at the single-clone level uncover cellular targets of interest in tumor-associated myeloid-derived suppressor cell populations.

Myeloid-derived suppressor cells (MDSCs) are immune cells that exert immunosuppression within the tumor, protecting cancer cells from the host's immune system and/or exogenous immunotherapies. While current research has been mostly focused in countering MDSC-driven immunosuppression, little is known about the mechanisms by which MDSCs disseminate/infiltrate cancerous tissue. This study looks into the use of microtextured surfaces, coupled with in vitro and in vivo cellular and molecular analysis tools, to videoscopically evaluate the dissemination patterns of MDSCs under structurally guided migration, at the single-cell level. MDSCs exhibited topographically driven migration, showing significant intra- and inter-population differences in motility, with velocities reaching ~40 µm h-1. Downstream analyses coupled with single-cell migration uncovered the presence of specific MDSC subpopulations with different degrees of tumor-infiltrating and anti-inflammatory capabilities. Granulocytic MDSCs showed a ~≥3-fold increase in maximum dissemination velocities and traveled distances, and a ~10-fold difference in the expression of pro- and anti-inflammatory markers. Prolonged culture also revealed that purified subpopulations of MDSCs exhibit remarkable plasticity, with homogeneous/sorted subpopulations giving rise to heterogenous cultures that represented the entire hierarchy of MDSC phenotypes within 7 days. These studies point towards the granulocytic subtype as a potential cellular target of interest given their superior dissemination ability and enhanced anti-inflammatory activity.

Stabilized collagen matrix dressing improves wound macrophage function and epithelialization.

Decellularized matrices of biologic tissue have performed well as wound care dressings. Extracellular matrix-based dressings are subject to rapid degradation by excessive protease activity at the wound environment. Stabilized, acellular, equine pericardial collagen matrix (sPCM) wound care dressing is flexible cross-linked proteolytic enzyme degradation resistant. sPCM was structurally characterized utilizing scanning electron and atomic force microscopy. In murine excisional wounds, sPCM was effective in mounting an acute inflammatory response. Postwound inflammation resolved rapidly, as indicated by elevated levels of IL-10, arginase-1, and VEGF, and lowering of IL-1ß and TNF-α. sPCM induced antimicrobial proteins S100A9 and ß-defensin-1 in keratinocytes. Adherence of Pseudomonas aeruginosa and Staphylococcus aureus on sPCM pre-exposed to host immune cells in vivo was inhibited. Excisional wounds dressed with sPCM showed complete closure at d 14, while control wounds remained open. sPCM accelerated wound re-epithelialization. sPCM not only accelerated wound closure but also improved the quality of healing by increased collagen deposition and maturation. Thus, sPCM is capable of presenting scaffold functionality during the course of wound healing. In addition to inducing endogenous antimicrobial defense systems, the dressing itself has properties that minimize biofilm formation. It mounts robust inflammation, a process that rapidly resolves, making way for wound healing to advance.-El Masry, M. S., Chaffee, S., Das Ghatak, P., Mathew-Steiner, S. S., Das, A., Higuita-Castro, N., Roy, S., Anani, R. A., Sen, C. K. Stabilized collagen matrix dressing improves wound macrophage function and epithelialization.

Lab-on-a-Chip Platforms for Biophysical Studies of Cancer with Single-Cell Resolution.

Recent cancer research has more strongly emphasized the biophysical aspects of tumor development, progression, and microenvironment. In addition to genetic modifications and mutations in cancer cells, it is now well accepted that the physical properties of cancer cells such as stiffness, electrical impedance, and refractive index vary with tumor progression and can identify a malignant phenotype. Moreover, cancer heterogeneity renders population-based characterization techniques inadequate, as individual cellular features are lost in the average. Hence, platforms for fast and accurate characterization of biophysical properties of cancer cells at the single-cell level are required. Here, we highlight some of the recent advances in the field of cancer biophysics and the development of lab-on-a-chip platforms for single-cell biophysical analyses of cancer cells.

Microfabricated mimics of in vivo structural cues for the study of guided tumor cell migration.

Guided cell migration plays a crucial role in tumor metastasis, which is considered to be the major cause of death in cancer patients. Such behavior is regulated in part by micro/nanoscale topographical cues present in the parenchyma or stroma in the form of fiber-like and/or conduit-like structures (e.g., white matter tracts, blood/lymphatic vessels, subpial and subperitoneal spaces). In this paper we used soft lithography micromolding to develop a tissue culture polystyrene platform with a microscale surface pattern that was able to induce guided cell motility along/through fiber-/conduit-like structures. The migratory behaviors of primary (glioma) and metastatic (lung and colon) tumors excised from the brain were monitored via time-lapse microscopy at the single cell level. All the tumor cells exhibited axially persistent cell migration, with percentages of unidirectionally motile cells of 84.0 ± 3.5%, 58.3 ± 6.8% and 69.4 ± 5.4% for the glioma, lung, and colon tumor cells, respectively. Lung tumor cells showed the highest migratory velocities (41.8 ± 4.6 µm h(-1)) compared to glioma (24.0 ± 1.8 µm h(-1)) and colon (26.7 ± 2.8 µm h(-1)) tumor cells. This platform could potentially be used in conjunction with other biological assays to probe the mechanisms underlying the metastatic phenotype under guided cell migration conditions, and possibly by itself as an indicator of the effectiveness of treatments that target specific tumor cell motility behaviors.

miR-146a regulates mechanotransduction and pressure-induced inflammation in small airway epithelium.

Mechanical ventilation generates biophysical forces, including high transmural pressures, which exacerbate lung inflammation. This study sought to determine whether microRNAs (miRNAs) respond to this mechanical force and play a role in regulating mechanically induced inflammation. Primary human small airway epithelial cells (HSAEpCs) were exposed to 12 h of oscillatory pressure and/or the proinflammatory cytokine TNF-α. Experiments were also conducted after manipulating miRNA expression and silencing the transcription factor NF-κB or toll-like receptor proteins IRAK1 and TRAF6. NF-κB activation, IL-6/IL-8/IL-1ß cytokine secretion, miRNA expression, and IRAK1/TRAF6 protein levels were monitored. A total of 12 h of oscillatory pressure and TNF-α resulted in a 5- to 7-fold increase in IL-6/IL-8 cytokine secretion, and oscillatory pressure also resulted in a time-dependent increase in IL-6/IL-8/IL-1ß cytokine secretion. Pressure and TNF-α also resulted in distinct patterns of miRNA expression, with miR-146a being the most deregulated miRNA. Manipulating miR-146a expression altered pressure-induced cytokine secretion. Silencing of IRAK1 or TRAF6, confirmed targets of miR-146a, resulted in a 3-fold decrease in pressure-induced cytokine secretion. Cotransfection experiments demonstrate that miR-146a's regulation of pressure-induced cytokine secretion depends on its targeting of both IRAK1 and TRAF6. MiR-146a is a mechanosensitive miRNA that is rapidly up-regulated by oscillatory pressure and plays an important role in regulating mechanically induced inflammation in lung epithelia.

Vacuum-assisted cell seeding in a microwell cell culture system.

We present a simple method to actively pattern individual cells and groups of cells in a polymer-based microdevice using vacuum-assisted cell seeding. Soft lithography is used to mold polymer microwells with various geometries on top of commercially available porous membranes. Cell suspensions are placed in a vacuum filtration setup to pull culture medium through the microdevice, trapping the cells in the microwells. The process is evaluated by determining the number of cells per microwell for a given cell seeding density and microwell geometry. This method is tested with adherent and nonadherent cells (NIH 3T3 fibroblasts, PANC-1 pancreatic ductal epithelial-like cells, and THP-1 monocytic leukemia cells). These devices could find applications in high-throughput cell screening, cell transport studies, guided formation of cell clusters, and tissue engineering.

ISOLATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS AND EVALUATION OF THEIR OSTEOGENIC POTENTIAL

Human bone marrow mesenchymal stem cells (hBMSCs) comprise a cell population capable of self-renewal and multilineage differentiation commonly isolated from bone marrow aspirates of large bones. Their osteogenic potential has been extensively exploited for the biological evaluation of scaffolds or biomaterials with applications in bone tissue engineering. This work aimed to isolate hBMSCs from femoral heads of patients undergoing total hip arthroplasty and to evaluate their osteogenic potential. Briefly, the trabecular bone was extracted and mechanically disaggregated; the released cells were cultured and non-adherent cells were removed after 4 days. The osteogenic potential was evaluated at the fifth passage after 14 and 20 days of induction, comparing cultures with and without osteogenic supplements, via Alizarin red staining and the quantification of the gene expression levels of the osteogenic markers collagen type I, osteonectin and bone sialoprotein through real-time RT-PCR. The obtained hBMSCs presented a stable undifferentiated phenotype after prolonged cell culture, matrix mineralization capabilities and expression of osteoblast phenotype upon osteogenic induction. The three markers were up-regulated in cultures under osteogenic conditions and 2 fold differences in their expression levels were found to be significant for the onset of the differentiation process. The obtained hBMSCs may have applications on the in vitro evaluation of the osteoinductivity of different biomaterials, bioactive molecules or tissue engineering scaffolds.

Las células madre mesenquimatosas de médula ósea humana (abreviadas hBMSCs) constituyen una fuente de células auto-renovables con alto potencial de diferenciación, comúnmente aisladas a partir de los aspirados medulares en huesos largos. Su diferenciación hacia el linaje osteogénico, por ejemplo, ha sido ampliamente utilizada para la evaluación biológica de biomateriales o matrices con aplicaciones en la ingeniería de tejidos óseos. El objetivo de este trabajo consistió en aislar hBMSCs a partir de la cabeza femoral de pacientes sometidos a artroplastia total de cadera, así como evaluar su potencial osteogénico. Brevemente, se extrajo el hueso esponjoso y se disgregó mecánicamente; las células desprendidas se cultivaron y las células no adherentes se eliminaron luego de 4 días. El potencial osteogénico se evaluó en la quinta generación de cultivo, mediante ensayos de diferenciación a 14 y 20 días donde se compararon cultivos con y sin suplementos osteogénicos. La evaluación se realizó mediante tinción con Alizarina Roja y la cuantificación de los niveles de expresión génica de los marcadores osteogénicos colágeno tipo I, osteonectinca y sialoprotiena ósea mediante RT-PCR en tiempo real. Las hBMSCs obtenidas presentaron un fenotipo no-diferenciado estable, así como la capacidad de mineralizar la matriz extracelular y expresar un fenotipo similar al osteoblasto durante la inducción osteogénica. Los tres marcadores evaluados se sobre-expresaron en los cultivos en condiciones osteogénicas, y se encontró que cambios hasta de 2X en sus niveles de expresión son relevantes para el desarrollo del proceso de diferenciación. El modelo de hBMSCS presentado podría ser utilizado para la evaluación in vitro de la osteoinductividad de diferentes biomateriales, moléculas bioactivas o matrices para ingeniería de tejidos.

MECHANICAL CHARACTERIZATION OF CELLS AND TISSUES AT THE MICRO SCALE

Cell-substrate interactions are relevant for a number of biological and clinical applications e.g. to determine the effectiveness of medical implants. Cells are natural transducers that respond to and sense signals originating in their microenvironment. One important cell signaling mechanism is known as chemo-mechanical transduction. This refers to the use of external mechanical cues to initiate internal biochemical cellular processes and vice versa. One key factor to characterize and understand these interactions is the evaluation of the mechanical forces present at the cell-substrate interface. Recent advances in the micro and nanotechnology fields have allowed the development of new tools for the measurement of cellular and tissue forces. These tools have provided a means to study extremely low cellular and subcellular forces (pN-µN) as well as detailed small-scale tissue mechanics. This paper will review some of the most significant approaches to characterize the mechanical properties of cells and tissues at the micro-scale. Material properties, device fabrication, and design issues will be discussed.

Las interacciones célula-sustrato juegan un papel fundamental en gran número de aplicaciones biológicas y clínicas. Las células son transductores naturales que sensan y responden a señales en su entorno fisiológico. Uno de los mecanismos más importantes empleados en la caracterización de interacciones celulares es la transducción químico-mecánica, la cual se basa en la implementación de señales externas que se aplican a la célula con el fin de inducir diversos procesos bioquímicos al interior de ésta y viceversa. Los avances alcanzados en el campo de la micro y nanotecnología han permitido el desarrollo de nuevas herramientas para medir fuerzas a nivel celular o incluso sub-celular (pN-µN), y dilucidar la mecánica de los tejidos en la escala micrométrica. La presente revisión literaria describe algunos de los micro-dispositivos empleados actualmente para caracterizar las propiedades mecánicas de las células y tejidos en la micro-escala.

Diseño y construcción del prototipo de una bomba de infusión tipo PAC para la administración de medicamentos / Design and construction of an infusion pump PCA (patient controlled analgesia) for the administration of medications

El presente artículo contiene los resultados del proyecto de diseño y construcción de un prototipo de Bomba de Infusión tipo PCA (analgesia controlada por el paciente) , para la administración de medicamentos, realizado como trabajo de grado para optar al titulo de Ingeniería Biomédica de la Escuela de Ingeniería de Antioquia - EIA y la Universidad CES. El prototipo desarrollado permite la administración por vía intravenosa de medicamentos, principalmente analgésicos para el tratamiento del dolor, con previa programación de parámetros tales como el volumen de la dosis y la rata de infusión; adicionalmente permite a los pacientes auto-administrarse dosis extras, mediante el uso de un pulsador y visualizar en computador los datos programados...