Non-viral Gene Delivery Methods for Bone and Joints.

Viral carrier transport efficiency of gene delivery is high, depending on the type of vector. However, viral delivery poses significant safety concerns such as inefficient/unpredictable reprogramming outcomes, genomic integration, as well as unwarranted immune responses and toxicity. Thus, non-viral gene delivery methods are more feasible for translation as these allow safer delivery of genes and can modulate gene expression transiently both in vivo, ex vivo, and in vitro. Based on current studies, the efficiency of these technologies appears to be more limited, but they are appealing for clinical translation. This review presents a summary of recent advancements in orthopedics, where primarily bone and joints from the musculoskeletal apparatus were targeted. In connective tissues, which are known to have a poor healing capacity, and have a relatively low cell-density, i.e., articular cartilage, bone, and the intervertebral disk (IVD) several approaches have recently been undertaken. We provide a brief overview of the existing technologies, using nano-spheres/engineered vesicles, lipofection, and in vivo electroporation. Here, delivery for microRNA (miRNA), and silencing RNA (siRNA) and DNA plasmids will be discussed. Recent studies will be summarized that aimed to improve regeneration of these tissues, involving the delivery of bone morphogenic proteins (BMPs), such as BMP2 for improvement of bone healing. For articular cartilage/osteochondral junction, non-viral methods concentrate on targeted delivery to chondrocytes or MSCs for tissue engineering-based approaches. For the IVD, growth factors such as GDF5 or GDF6 or developmental transcription factors such as Brachyury or FOXF1 seem to be of high clinical interest. However, the most efficient method of gene transfer is still elusive, as several preclinical studies have reported many different non-viral methods and clinical translation of these techniques still needs to be validated. Here we discuss the non-viral methods applied for bone and joint and propose methods that can be promising in clinical use

Isolation of human bone marrow mesenchymal stem cells and evaluation of their osteogenic potential

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.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

Neurogenic Tissue Nanotransfection in the Management of Cutaneous Diabetic Polyneuropathy

This work rests on our recent report on the successful use of tissue nanotransfection (TNT) delivery of Ascl1, Brn2, and Myt1l (TNTABM) to directly convert skin fibroblasts into electrophysiologically active induced neuronal cells (iN) in vivo. Here we report that in addition to successful neurogenic conversion of cells, TNTABM caused neurotrophic enrichment of the skin stroma. Thus, we asked whether such neurotrophic milieu of the skin can be leveraged to rescue pre-existing nerve fibers under chronic diabetic conditions. Topical cutaneous TNTABM caused elevation of endogenous NGF and other co-regulated neurotrophic factors such as Nt3. TNTABM spared loss of cutaneous PGP9.5+ mature nerve fibers in db/db diabetic mice. This is the first study demonstrating that under conditions of in vivo reprogramming, changes in the tissue microenvironment can be leveraged for therapeutic purposes such as the rescue of pre-existing nerve fibers from its predictable path of loss under conditions of diabetes

Nanotransfection-based vasculogenic cell reprogramming drives functional recovery in a mouse model of ischemic stroke

Ischemic stroke causes vascular and neuronal tissue deficiencies that could lead to substantial functional impairment and/or death. Although progenitor-based vasculogenic cell therapies have shown promise as a potential rescue strategy following ischemic stroke, current approaches face major hurdles. Here, we used fibroblasts nanotransfected with Etv2, Foxc2, and Fli1 (EFF) to drive reprogramming-based vasculogenesis, intracranially, as a potential therapy for ischemic stroke. Perfusion analyses suggest that intracranial delivery of EFF-nanotransfected fibroblasts led to a dose-dependent increase in perfusion 14 days after injection. MRI and behavioral tests revealed ~70% infarct resolution and up to ~90% motor recovery for mice treated with EFF-nanotransfected fibroblasts. Immunohistological analysis confirmed increases in vascularity and neuronal cellularity, as well as reduced glial scar formation in response to treatment with EFF-nanotransfected fibroblasts. Together, our results suggest that vasculogenic cell therapies based on nanotransfection-driven (i.e., nonviral) cellular reprogramming represent a promising strategy for the treatment of ischemic stroke

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 PCA para la administración de medicamentos

145 páginasEl presente proyecto describe el proceso de diseño y construcción de un prototipo de bomba de infusión tipo PCA. Éste equipo médico permite la administración por vía intravenosa de medicamentos, principalmente analgésicos para el tratamiento del dolor, con previa programación del volumen de la dosis que se le debe administrar al paciente y el tiempo de separación entre dosis sucesivas. Las características de precisión, seguridad y regularidad que brindan estos equipos en la administración del medicamento, hacen de él un equipo de uso indispensable en el ambiente hospitalario. El diseño funcional y estructural del prototipo y los materiales empleados para su construcción se determinaron a partir del análisis de la información recopilada a través de una revisión de la literatura, disponible en textos, manuales de bombas de infusión, cartillas para el control del dolor, múltiples sitios de Internet a partir de diferentes motores de búsqueda, encuestas y entrevistas realizadas al personal médico y de enfermería que trabaja con este tipo de equipos en diferentes instituciones médicas del departamento de Antioquia. El prototipo construido es una bomba de infusión PCA (analgesia controlada por el paciente) tipo jeringa, que permite la infusión de medicamentos de tres formas diferentes: continua, en la cual el paciente recibe periódicamente dosis de medicamento; PCA (analgesia controlada por el paciente), en la cual el paciente, mediante un pulsador, se auto-administra dosis de medicamento según la intensidad de su dolor, con restricciones que evitan sobredosis; y una forma combinada en la cual el paciente recibe continuamente dosis de medicamento y al mismo tiempo se puede auto-administrar dosis extras cuando tiene un dolor muy alto. Adicionalmente, permite el envió de datos, tales como hora, fecha, volúmenes infundidos y tiempo entre dosis a un computador. Cuenta además, con un sistema de alarmas que alerta de problemas en el funcionamiento. Mediante un monitoreo se verificó el correcto funcionamiento del prototipo. Los resultados obtenidos en este monitoreo permiten afirmar que es viable el desarrollo de bombas de infusión a nivel nacional, lo que generaría una reducción de costos y progreso al país.ABSTRACT: The present project describes the design process and construction of a prototype of PCA infusion pump. This medical equip allows the administration of medications via intravenous, mainly analgesic for the treatment of the pain, with previous programming of the volume of the dose that should be administered at the patient and the time of separation among successive dose. The characteristics of precision, security and regularity that offer these teams in the administration of the medication make of him an equipment of indispensable use in the hospital atmosphere. The functional and structural design of the prototype and the materials employees for their construction were determined starting from the analysis of the information gathered through a revision of the literature, available in texts, manuals of infusion bombs, notes for the control of the pain, multiple places of Internet starting from different search motors, surveys and interviews carried out the medical personnel and of infirmary that he/she works with this type of teams in different medical institutions of the department of Antioquia. The built prototype is an infusion bomb PCA (analgesia controlled by the patient) type squirts that allows the infusion of medications in three different ways: continuous, in which the patient receives medication dose periodically; PCA (analgesia controlled by the patient), in the one which the patient, by means of a push button, medication dose is car-administered according to the intensity of its pain, with restrictions that avoid overdose; and a form combined in which the patient receives medication dose continually and at the same time she can car-administer himself extra dose when he has a very high pain. Additionally, it allows the one he/she sent of data, such as hour, it dates, infused volumes and time among dose to a computer to store them. It also counts, with a system of alarms that alerts of problems in the operation. By means of a periodic registration carried out during two weeks the correct operation of the prototype it was verified. The results obtained in this registration allow affirming that it is viable the development of infusion bombs at national level, generating a reduction of costs and progress to the country.PregradoIngeniero(a) Biomédico(a

Diseño y construcción del prototipo de una bomba de infusión tipo PCA para la administración de medicamentos

The present article contains the results of the design and construction of an Infusion Pump PCA (patient controlled analgesia) prototype project, for the administration of medications, made as grade work to opt the Title of Biomedical Engineering of the EIA and CES Universities. The developed prototype allows the administration via intravenous of medications, mainly analgesic for treatment of pain, with previous programming parameters such as volume of the dose and the infusion rat; additionally it allows to the patient self~administer extra dose, by means of a pusher and to visualize in computer the scheduled data.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 título de Ingeniera Biomédica de la Escuela de Ingeniería de Antioquia - EIA Y el Instituto de Ciencias de la Salud – 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