A Micro-scale Humanized Ventilator-on-a-Chip to Examine the Injurious Effects of Mechanical Ventilation.

Patients with compromised respiratory function frequently require mechanical ventilation to survive. Unfortunately, non-uniform ventilation of injured lungs generates complex mechanical forces that lead to ventilator induced lung injury (VILI). Although investigators have developed lung-on-a-chip systems to simulate normal respiration, modeling the complex mechanics of VILI as well as the subsequent recovery phase is a challenge. Here we present a novel humanized in vitro ventilator-on-a-chip (VOC) model of the lung microenvironment that simulates the different types of injurious forces generated in the lung during mechanical ventilation. We used transepithelial/endothelial electrical resistance (TEER) measurements to investigate how individual and simultaneous application of the different mechanical forces alters real-time changes in barrier integrity during and after injury. We find that compressive stress (i.e. barotrauma) does not significantly alter barrier integrity while over-distention (20% cyclic radial strain, volutrauma) results in decreased barrier integrity that quickly recovers upon removal of mechanical stress. Conversely, surface tension forces generated during airway reopening (atelectrauma), result in a rapid loss of barrier integrity with a delayed recovery relative to volutrauma. Simultaneous application of cyclic stretching (volutrauma) and airway reopening (atelectrauma), indicate that the surface tension forces associated with reopening fluid-occluded lung regions is the primary driver of barrier disruption. Thus, our novel VOC system can monitor the effects of different types of injurious forces on barrier disruption and recovery in real-time and can be used to identify the biomechanical mechanisms of VILI.

A micro-scale humanized ventilator-on-a-chip to examine the injurious effects of mechanical ventilation.

Patients with compromised respiratory function frequently require mechanical ventilation to survive. Unfortunately, non-uniform ventilation of injured lungs generates complex mechanical forces that lead to ventilator induced lung injury (VILI). Although investigators have developed lung-on-a-chip systems to simulate normal respiration, modeling the complex mechanics of VILI as well as the subsequent recovery phase is a challenge. Here we present a novel humanized in vitro ventilator-on-a-chip (VOC) model of the lung microenvironment that simulates the different types of injurious forces generated in the lung during mechanical ventilation. We used transepithelial/endothelial electrical impedance measurements to investigate how individual and simultaneous application of mechanical forces alters real-time changes in barrier integrity during and after injury. We find that compressive stress (i.e. barotrauma) does not significantly alter barrier integrity while over-distention (20% cyclic radial strain, volutrauma) results in decreased barrier integrity that quickly recovers upon removal of mechanical stress. Conversely, surface tension forces generated during airway reopening (atelectrauma), result in a rapid loss of barrier integrity with a delayed recovery relative to volutrauma. Simultaneous application of cyclic stretching (volutrauma) and airway reopening (atelectrauma), indicates that the surface tension forces associated with reopening fluid-occluded lung regions are the primary driver of barrier disruption. Thus, our novel VOC system can monitor the effects of different types of injurious forces on barrier disruption and recovery in real-time and can be used to interogate the biomechanical mechanisms of VILI.

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.

Engineered extracellular vesicles from human skin cells induce pro-ß-cell conversions in pancreatic ductal cells.

Direct nuclear reprogramming has the potential to enable the development of ß cell replacement therapies for diabetes that do not require the use of progenitor/stem cell populations. However, despite their promise, current approaches to ß cell-directed reprogramming rely heavily on the use of viral vectors. Here we explored the use of extracellular vesicles (EVs) derived from human dermal fibroblasts (HDFs) as novel non-viral carriers of endocrine cell-patterning transcription factors, to transfect and transdifferentiate pancreatic ductal epithelial cells (PDCs) into hormone-expressing cells. Electrotransfection of HDFs with expression plasmids for Pdx1, Ngn3, and MafA (PNM) led to the release of EVs loaded with PNM at the gene, mRNA, and protein level. Exposing PDC cultures to PNM-loaded EVs led to successful transfection and increased PNM expression in PDCs, which ultimately resulted in endocrine cell-directed conversions based on the expression of insulin/c-peptide, glucagon, and glucose transporter 2 (Glut2). These findings were further corroborated in vivo in a mouse model following intraductal injection of PNM- vs sham-loaded EVs. Collectively these findings suggest that dermal fibroblast-derived EVs could potentially serve as a powerful platform technology for the development and deployment of non-viral reprogramming-based cell therapies for insulin-dependent diabetes.

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.

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.

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...