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An association between body mass index (BMI; weight (kg)/height (m)2) and low back pain (LBP) has long been debated, but inconsistent measurements of BMI and varying definitions of LBP have produced conflicting findings. We explored this association using measured BMI and physician documentation of recurrent LBP among healthy young adults. Data were extracted from the Israel Defense Forces electronic medical record system. All Israeli citizens with compulsory military service during January 2008-March 2019 were included (n = 705,840). Exclusion criteria were spine deformities, disc pathologies, spinal surgeries, arthropathies, connective tissue diseases, pain syndromes, low bone density disorders, cancers, and psychiatric illnesses. LBP was defined as electronic medical record system documentation of 1) 2 medical visits at least 6 weeks apart with a diagnosis of LBP or "LBP with radiation" or 2) 1 medical visit resulting in referral to an orthopedic surgeon. Logistic regression models were used to explore the association between BMI category and LBP; 619,969 (87.8%) individuals (mean age = 18.9 (standard deviation, 0.97) years; 56.9% male) were included. LBP prevalence was 9.2% (n = 56,918) and higher among males (9.7%) than females (8.5%). Overweight (odds ratio = 1.123, 95% confidence interval: 1.096, 1.151) and obesity (odds ratio = 1.137, 95% confidence interval: 1.096, 1.179) were associated with LBP. The association remained significant after accounting for various sociodemographic factors. Maintaining a healthy BMI may aid in the prevention of LBP in young adults.
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Dolor de la Región Lumbar , Femenino , Humanos , Masculino , Adulto Joven , Adolescente , Dolor de la Región Lumbar/epidemiología , Dolor de la Región Lumbar/etiología , Índice de Masa Corporal , Obesidad/epidemiología , Obesidad/complicaciones , Sobrepeso/complicaciones , CausalidadRESUMEN
BACKGROUND: Medical organizations worldwide aim for equity and diversity in the medical profession to improve care quality. Data on whether the caregiver gender affects outcomes in the prehospital setting are essential but scarce compared to available in-hospital studies. OBJECTIVES: To analyze the rates of missed injuries in the prehospital setting and determine whether these rates were associated with the gender of the on-field physician or paramedic. METHODS: A retrospective record review was conducted, which included trauma records documented in two trauma registries, the prehospital Israel Defense Forces-Trauma Registry (IDF-TR), and the in-hospital Israeli National Trauma Registry (INTR). Missed injuries were defined as injuries documented in the INTR but not in the IDF-TR. A multivariable regression analysis was performed to assess the association between provider's gender and missed injuries. RESULTS: Of 490 casualties, 369 (75.3%) were treated by teams that included only male paramedics or physicians. In 386 (78.8%) cases, a physician was a part of the prehospital team. In all, 94 (19.2%) casualties sustained injuries that were missed by the prehospital medical team. Missed injuries were not associated with the gender of the paramedic or physician (odds ratio 1.242, 95% confidence interval 0.69-2.193). CONCLUSIONS: No association was found between the gender of the medical provider in the prehospital setting and the rate of missed injuries. These results should encourage prehospital emergency medical systems to aim for a balanced and diverse caregiver population.
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Servicios Médicos de Urgencia , Heridas y Lesiones , Servicios Médicos de Urgencia/métodos , Humanos , Israel/epidemiología , Masculino , Sistema de Registros , Estudios Retrospectivos , Heridas y Lesiones/diagnóstico , Heridas y Lesiones/epidemiología , Heridas y Lesiones/terapiaRESUMEN
INTRODUCTION: Pediatric coronal plane knee deformities can be treated surgically using hemiepiphysiodesis. The two leading techniques used for hemiepiphysiodesis are: tension-band plates (TBP) and percutaneous transphyseal screws (PETS). We hypothesized that PETS would lead to faster guided correction of angular knee deformities than TBP. MATERIALS AND METHODS: A retrospective cohort of 35 patients treated with either TBP or PETS in one medical institution was established. The cohort included both genu varum and genu valgum of both primary and secondary etiologies. We first compared the treatment groups for differences in demographic and malalignment characteristics. Then, we compared the treatment groups for differences in operation-related outcomes, radiological mechanical correction and complication rates. RESULTS: We found that the use of PETS, compared to TBP, was associated with a faster implantation surgery and a shorter interval between implantation and removal, i.e., faster correction. Furthermore, PETS were associated with faster correction rates of the mechanical axis deviation, lateral distal femoral angle and medial proximal tibial angle. No significant differences in complication rates were found between the two treatments. CONCLUSION: PETS provided a faster correction of angular knee deformities compared to TBP at similar complication rates. Hence, PETS could be considered a superior technique for hemiepiphysiodesis.
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Genu Valgum , Genu Varum , Placas Óseas , Tornillos Óseos , Niño , Genu Valgum/cirugía , Humanos , Estudios RetrospectivosRESUMEN
BACKGROUND: Endoscopic retrograde cholangiopancreatography (ERCP) is essential for managing biliary and pancreatic disorders. Infection is the most morbid complication of ERCP and among the most common causes of ERCP-related death. CASE PRESENTATION: A 69-year-old man presented with right upper quadrant abdominal pain, obstructive jaundice and abnormal liver function tests. Ultrasound revealed cholelithiasis without bile duct dilation. After receiving intravenous antibiotics for acute cholecystitis, the patient was discharged. Two weeks later, an endoscopic ultrasound demonstrated gallstones and CBD dilation of up to 6.4 mm with 2 filling defects. An ERCP was performed with a papillotomy and stone extraction. Twenty-four hours post-ERCP the patient developed a fever, chills, bilirubinemia and elevated liver function tests. Ascending cholangitis was empirically treated using Ceftriaxone and Metronidazole. However, the patient remained febrile, with a diffusely tender abdomen and elevated inflammatory markers. A CT revealed a very small hypodense lesion in the seventh liver segment. Extended-spectrum beta-lactamase positive Klebsiella Pneumonia and Enterococcus Hirae were identified, and the antibiotics were switched to Imipenem and Cilastatin. The hypodense lesion in the liver increased to 1.85 cm and a new hypodense lesion was seen in the right psoas. At day 10 post-ERCP, the patient started having low back pain and difficulty walking. MRI revealed L4-L5 discitis with a large epidural abscess, spanning L1-S1 and compressing the spinal cord. Decompressive laminectomy of L5 was done and Klebsiella pneumonia was identified. Due to continued drainage from the wound, high fever, we performed a total body CT which revealed increased liver and iliopsoas abscess. Decompressive laminectomy was expanded to include L2-L4 and multiple irrigations were done. Gentamycin and Vancomycin containing polymethylmethacrylate beads were implanted locally and drainage catheters were placed before wound closure. Multidisciplinary panel discussion was performed, and it was decided to continue with a non invasive approach . CONCLUSIONS: Early recognition of complications and individualized therapy by a multi-disciplined team is important for managing post-ERCP septic complications. Particular attention should be given to adequate coverage by empiric antibiotics.
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Absceso/etiología , Colangiopancreatografia Retrógrada Endoscópica/métodos , Colangitis/diagnóstico , Anciano , Endosonografía , Cálculos Biliares/diagnóstico , Humanos , Ictericia Obstructiva/etiología , Imagen por Resonancia Magnética , Masculino , UltrasonografíaRESUMEN
Ligament injuries occur frequently, substantially hindering routine daily activities and sports participation in patients. Surgical reconstruction using autogenous or allogeneic tissues is the gold standard treatment for ligament injuries. Although surgeons routinely perform ligament reconstructions, the integrity of these reconstructions largely depends on adequate biological healing of the interface between the ligament graft and the bone. We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would lead to significantly improved ligament graft integration. To test this hypothesis, an anterior cruciate ligament reconstruction procedure was performed in Yucatan mini-pigs. A collagen scaffold was implanted in the reconstruction sites to facilitate recruitment of endogenous mesenchymal stem cells. Ultrasound-mediated reporter gene delivery successfully transfected 40% of cells recruited to the reconstruction sites. When BMP-6 encoding DNA was delivered, BMP-6 expression in the reconstruction sites was significantly enhanced. Micro-computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to significantly enhanced osteointegration in all animals 8 weeks after surgery. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchymal progenitor cells can effectively improve ligament reconstruction in large animals, thereby addressing a major unmet orthopedic need and offering new possibilities for translation to the clinical setting.
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Aloinjertos/citología , Reconstrucción del Ligamento Cruzado Anterior/métodos , Ligamentos/citología , Tendones/citología , Aloinjertos/metabolismo , Animales , Proteína Morfogenética Ósea 6/metabolismo , Colágeno/metabolismo , Técnicas de Transferencia de Gen , Ligamentos/metabolismo , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Células Madre/citología , Células Madre/metabolismo , Porcinos , Porcinos Enanos , Tendones/metabolismo , Trasplante Homólogo/métodos , Ultrasonografía/métodos , Microtomografía por Rayos X/métodosRESUMEN
PURPOSE OF REVIEW: The purpose of this review is to discuss the recent advances in gene therapy as a treatment for bone regeneration. While most fractures heal spontaneously, patients who present with fracture nonunion suffer from prolonged pain, disability, and often require additional operations to regain musculoskeletal function. RECENT FINDINGS: In the last few years, BMP gene delivery by means of electroporation and sonoporation resulted in repair of nonunion bone defects in mice, rats, and minipigs. Ex vivo transfection of porcine mesenchymal stem cells (MSCs) resulted in bone regeneration following implantation in vertebral defects of minipigs. Sustained release of VEGF gene from a collagen-hydroxyapatite scaffold to the mandible of a human patient was shown to be safe and osteoinductive. In conclusion, gene therapy methods for bone regeneration are systematically becoming more efficient and show proof-of-concept in clinically relevant animal models. Yet, on the pathway to clinical use, more investigation is needed to determine the safety aspects of the various techniques in terms of biodistribution, toxicity, and tumorigenicity.
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Proteínas Morfogenéticas Óseas/genética , Regeneración Ósea/genética , Curación de Fractura/genética , Fracturas no Consolidadas/terapia , Terapia Genética/métodos , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Colágeno , Durapatita , Electroporación , Humanos , Trasplante de Células Madre Mesenquimatosas , Andamios del Tejido , TransfecciónRESUMEN
Increased counts and priming of peripheral polymorphonuclear leukocytes (PMNLs) are associated with future or ongoing atherosclerosis; however, the role of PMNLs in enhancing monocyte transendothelial migration is still unclear. Our aims were to examine endothelial and monocyte activation, transmigration, and posttransmigration activation induced ex vivo by in vivo primed PMNLs and the effect of antioxidants on the activation. A unique ex vivo coculture system of three cell types was developed in this study, enabling interactions among the following: primary human umbilical vein endothelial cells (HUVECs), monocytes (THP-1 cell line), and in vivo primed PMNLs from hemodialysis (HD) patients and healthy control (HC) subjects. The interactions among these cells were examined, and an intervention with superoxide dismutase and catalase was performed. Preexposed HUVECs to HD/HC PMNLs showed a significant monocyte transmigration yield, 120-170% above HCs. Monocyte exposure to HD PMNLs induced pre- and posttransmigration activation. When the three cell types were cocultivated at the same time, monocyte chemoattractant protein-1 protein levels released from HUVECs, and activation markers on HUVECs [CD54 and chemokine (C-X3-C motif) ligand 1] and monocytes [chemokine (C-X3-C) receptor 1 and chemokine (C-C motif) receptor 2] were increased. Monocyte transmigration yield decreased to 70% (compared with HC subjects) due to adherence and accumulation of monocytes to HUVECs. When superoxide dismutase and catalase were used, reduced HUVEC and monocyte activation markers brought the transmigration yields to control levels and abolished accumulation of monocytes, emphasizing the role of superoxide in this process. We conclude that peripheral primed PMNLs play a pivotal role in enhancing monocyte transendotelial migration, the hallmark of the atherosclerotic process. Primed PMNLs can be used as a mediator and a biomarker of atherosclerosis even before plaque formation.NEW & NOTEWORTHY Primed polymorphonuclear leukocytes are key mediators in monocyte transendothelial migration, a new understanding of the initiation of endothelial dysfunction and monocyte activation, transmigration, and accumulation in the subendothelial layer.
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Monocitos/fisiología , Neutrófilos/fisiología , Diálisis Renal , Migración Transendotelial y Transepitelial/fisiología , Aterosclerosis/patología , Catalasa/farmacología , Adhesión Celular/fisiología , Quimiocina CCL2 , Técnicas de Cocultivo , Endotelio Vascular , Células Endoteliales de la Vena Umbilical Humana , Humanos , Fallo Renal Crónico/sangre , Activación de Macrófagos , Superóxido Dismutasa/farmacologíaRESUMEN
Osteoporosis affects more than 200 million people worldwide leading to more than 2 million fractures in the United States alone. Unfortunately, surgical treatment is limited in patients with low bone mass. Parathyroid hormone (PTH) was shown to induce fracture repair in animals by activating mesenchymal stem cells (MSCs). However, it would be less effective in patients with fewer and/or dysfunctional MSCs due to aging and comorbidities. To address this, we evaluated the efficacy of combination i.v. MSC and PTH therapy versus monotherapy and untreated controls, in a rat model of osteoporotic vertebral bone defects. The results demonstrated that combination therapy significantly increased new bone formation versus monotherapies and no treatment by 2 weeks (P < 0.05). Mechanistically, we found that PTH significantly enhanced MSC migration to the lumbar region, where the MSCs differentiated into bone-forming cells. Finally, we used allogeneic porcine MSCs and observed similar findings in a clinically relevant minipig model of vertebral defects. Collectively, these results demonstrate that in addition to its anabolic effects, PTH functions as an adjuvant to i.v. MSC therapy by enhancing migration to heal bone loss. This systemic approach could be attractive for various fragility fractures, especially using allogeneic cells that do not require invasive tissue harvest.
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Regeneración Ósea/efectos de los fármacos , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/efectos de los fármacos , Osteoporosis/terapia , Hormona Paratiroidea/farmacología , Fracturas de la Columna Vertebral/terapia , Animales , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Terapia Combinada , Modelos Animales de Enfermedad , Femenino , Humanos , Células Madre Mesenquimatosas/citología , Osteoporosis/complicaciones , Ratas , Fracturas de la Columna Vertebral/etiología , PorcinosRESUMEN
INTRODUCTION: Promoting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine acceptance and uptake became necessary to achieve a high vaccination rate and subsequently herd immunity. Although the Israeli population has been largely acceptant of the SARS-CoV-2 vaccine, vaccine hesitancy has remained a major concern, especially in younger adults. We hypothesized that young adults who refused SARS-CoV-2 vaccination differed from those who have been adherent and could be characterized. Studying this specific population and recognizing individuals within this group who might be more probable to refuse vaccination can enable to target measures to further promote vaccination acceptance. METHODS: We conducted a cross-sectional comparison in a study population comprised of 17,435 Israeli Defense Forces (IDF) personnel who were SARS-CoV-2 vaccine eligible. This group included 14,834 vaccinated and 2,601 nonvaccinated individuals. Patient characteristics including occupational parameters, demographic features, psychotechnical grading (an intelligence assessment score), education level, and medical background were collected. RESULTS: The median age was 20.57 years and almost 80% were males. At the time of data collection, most individuals (85.1%, n = 14,834) have been vaccinated. Officers and noncommissioned officers were more likely to be vaccinated compared with regular soldiers (96%, and 90.2% vs. 83.3% respectively, P < .001), as well as combat battalions stationed personnel compared to their peers in rear and administrative units (89.4% vs. 78.4%, P < .001). Socioeconomic clusters were also associated with vaccination adherence, with 92.9% vs. 79.5% vaccination rates in the highest and lowest clusters, respectively (P < .001). Younger age, no previous immigration status, higher education level, and higher psychotechnical grades were also found associated with an increased likelihood of being vaccinated (P < .001). CONCLUSIONS: In a large cohort of enlisted IDF personnel, disparity in SARS-CoV-2 vaccine adherence was found to be related to multiple socioeconomic, educational, and military service-related variables. Although some differences were substantial, others were small and of questionable public health significance. Acknowledging these differences may enable community leaders, health care providers, and administrators to target specific populations in order to further promote SARS-CoV-2 vaccination acceptance.
RESUMEN
Osteopetrosis is a rare skeletal dysplasia resulting from an osteoclast defect leading to increased bone mass and density. Hematopoietic stem cell transplantation can rescue the disease phenotype and prevent complications. However, little is known about the skeletal changes hematopoietic stem cell transplantation induces in patients with this disease. The purpose of this study was to describe the skeletal changes after hematopoietic stem cell transplantation in a retrospective cohort of patients diagnosed with osteopetrosis in one medical center over 13 years. For this purpose, all available epidemiological, hematological, biochemical, and radiographic data were collected and quantitatively analyzed. We found a significant early change in bone metabolism markers coinciding with hematopoietic recovery after stem cell transplantation. Hematopoietic stem cell transplantation induced a later significant improvement in both skeletal mineral distribution and morphology but did not lead to complete radiological normalization. Presumably, changes in bone metabolism, skeletal mineral distribution, and morphology were the result of renewed osteoclast function enabling bone remodeling. We propose that biochemical bone metabolism markers and radiological indices be routinely used to evaluate response to hematopoietic stem cell transplantation in patients with osteopetrosis. © 2020 American Society for Bone and Mineral Research.
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Trasplante de Células Madre Hematopoyéticas , Osteopetrosis , Remodelación Ósea , Humanos , Osteoclastos , Osteopetrosis/diagnóstico por imagen , Osteopetrosis/terapia , Estudios RetrospectivosRESUMEN
Ultrasound-mediated gene delivery (sonoporation) is a minimally invasive, nonviral and clinically translatable method of gene therapy. This method offers a favorable safety profile over that of viral vectors and is less invasive as compared with other physical gene delivery approaches (e.g., electroporation). We have previously used sonoporation to overexpress transgenes in different skeletal tissues in order to induce tissue regeneration. Here, we provide a protocol that could easily be adapted to address various other targets of tissue regeneration or additional applications, such as cancer and neurodegenerative diseases. This protocol describes how to prepare, conduct and optimize ultrasound-mediated gene delivery in both a murine and a porcine animal model. The protocol includes the preparation of a microbubble-DNA mix and in vivo sonoporation under ultrasound imaging. Ultrasound-mediated gene delivery can be accomplished within 10 min. After DNA delivery, animals can be followed to monitor gene expression, protein secretion and other transgene-specific outcomes, including tissue regeneration. This procedure can be accomplished by a competent graduate student or technician with prior experience in ultrasound imaging or in performing in vivo procedures.
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ADN/genética , Técnicas de Transferencia de Gen/instrumentación , Neoplasias/terapia , Enfermedades Neurodegenerativas/terapia , Terapia por Ultrasonido/métodos , Ultrasonografía/métodos , Animales , ADN/metabolismo , Modelos Animales de Enfermedad , Terapia Genética/métodos , Humanos , Ratones , Microburbujas , Músculo Esquelético/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Plásmidos/química , Plásmidos/metabolismo , Porcinos , Porcinos Enanos , Ondas UltrasónicasRESUMEN
BACKGROUND: Polymorphonuclear leukocyte priming and low grade inflammation are related to severity of kidney disease. Erythropoietin-receptor is present on PMNLs. OBJECTIVESxi: To evaluate the effect of 20 weeks of epoetin-alpha treatment on PMNL characteristics in relation to the rate of kidney function deterioration in patients with chronic kidney disease. METHODS: Forty anemic chronic kidney disease patients, stage 4-5, were assigned to EPO and non-EPO treatment for 20 weeks. A group of 20 healthy controls was also studied. PMNL priming and PMNL-derived low grade inflammation were estimated, in vivo and ex vivo, before and after EPO treatment: The rate of superoxide release, white blood cells and PMNL counts, serum alkaline phosphatase and PMNL viability were measured. EPO-receptor on PMNLs was assayed by flow cytometry. The effect of 20 weeks of EPO treatment on kidney function was related to the estimated glomerular filtration rate. esults: EPO treatment attenuated superoxide release ex vivo and in vivo and promoted PMNL survival ex vivo. Decreased low grade inflammation was reflected by reduced WBC and PMNL counts and ALP activity following treatment. EPO retarded the deterioration in GFR. The percent of PMNLs expressing EPO-R was higher before EPO treatment and correlated positively with the rate of superoxide release. After 20 weeks of EPO treatment the percent of PMNLs expressing EPO-R was down-regulated. CONCLUSIONS: These non-erythropoietic properties of EPO are mediated by EPO-R on PMNLs, not related to the anemia correction. A new renal protection effect of EPO via attenuation of PMNL priming that decreases systemic low grade inflammation and oxidative stress is suggested.
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Eritropoyetina/uso terapéutico , Hematínicos/uso terapéutico , Fallo Renal Crónico/tratamiento farmacológico , Neutrófilos/efectos de los fármacos , Anciano , Anemia/complicaciones , Anemia/tratamiento farmacológico , Epoetina alfa , Eritropoyetina/sangre , Eritropoyetina/farmacología , Femenino , Tasa de Filtración Glomerular/efectos de los fármacos , Hematínicos/farmacología , Humanos , Fallo Renal Crónico/clasificación , Fallo Renal Crónico/metabolismo , Masculino , Neutrófilos/enzimología , Neutrófilos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Receptores de Eritropoyetina/efectos de los fármacos , Proteínas Recombinantes , Índice de Severidad de la Enfermedad , Superóxido Dismutasa/metabolismoRESUMEN
Intervertebral disc (IVD) degeneration is a leading cause of chronic low back pain that affects millions of people every year. Yet identification of the specific IVD causing this pain is based on qualitative visual interpretation rather than objective findings. One possible approach to diagnosing pain-associated IVD could be to identify acidic IVDs, as decreased pH within an IVD has been postulated to mediate discogenic pain. We hypothesized that quantitative chemical exchange saturation transfer (qCEST) MRI could detect pH changes in IVDs, and thence be used to diagnose pathologically painful IVDs objectively and noninvasively. To test this hypothesis, a surgical model of IVD degeneration in Yucatan minipigs was used. Direct measurement of pH inside the degenerated IVDs revealed a significant drop in pH after degeneration, which correlated with a significant increase in the qCEST signal. Gene analysis of harvested degenerated IVDs revealed significant upregulation of pain-, nerve- and inflammatory-related markers after IVD degeneration. A strong positive correlation was observed between the expression of pain markers and the increase in the qCEST signal. Collectively, these findings suggest that this approach might be used to identify which IVD is causing low back pain, thereby providing valuable guidance for pain and surgical management.
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Biomarcadores/metabolismo , Degeneración del Disco Intervertebral/metabolismo , Degeneración del Disco Intervertebral/fisiopatología , Disco Intervertebral/metabolismo , Disco Intervertebral/fisiopatología , Dolor de la Región Lumbar/metabolismo , Dolor de la Región Lumbar/fisiopatología , Animales , Modelos Animales de Enfermedad , Femenino , Concentración de Iones de Hidrógeno , Imagen por Resonancia Magnética/métodos , Porcinos , Porcinos EnanosRESUMEN
Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ages. Animal OVCF models are essential to the preclinical development of translational tissue engineering strategies. While a number of models currently exist, this protocol describes an optimized method for inducing multiple highly reproducible vertebral defects in a single nude rat. A novel longitudinal semiautomated microcomputed tomography (µCT)-based quantitative structural analysis of the vertebral defects is also detailed. Briefly, rats were imaged at multiple time points post-op. The day 1 scan was reoriented to a standard position, and a standard volume of interest was defined. Subsequent µCT scans of each rat were automatically registered to the day 1 scan so the same volume of interest was then analyzed to assess for new bone formation. This versatile approach can be adapted to a variety of other models where longitudinal imaging-based analysis could benefit from precise 3D semiautomated alignment. Taken together, this protocol describes a readily quantifiable and easily reproducible system for osteoporosis and bone research. The suggested protocol takes 4 months to induce osteoporosis in nude ovariectomized rats and between 2.7 and 4 h to generate, image, and analyze two vertebral defects, depending on tissue size and equipment.
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Osteoporosis/diagnóstico por imagen , Fracturas Osteoporóticas/diagnóstico por imagen , Fracturas de la Columna Vertebral/diagnóstico por imagen , Microtomografía por Rayos X/métodos , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Ratas , Ratas DesnudasRESUMEN
More than 2 million bone-grafting procedures are performed each year using autografts or allografts. However, both options carry disadvantages, and there remains a clear medical need for the development of new therapies for massive bone loss and fracture nonunions. We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would induce efficient bone regeneration and fracture repair. To test this hypothesis, we surgically created a critical-sized bone fracture in the tibiae of Yucatán mini-pigs, a clinically relevant large animal model. A collagen scaffold was implanted in the fracture to facilitate recruitment of endogenous mesenchymal stem/progenitor cells (MSCs) into the fracture site. Two weeks later, transcutaneous ultrasound-mediated reporter gene delivery successfully transfected 40% of cells at the fracture site, and flow cytometry showed that 80% of the transfected cells expressed MSC markers. Human bone morphogenetic protein-6 (BMP-6) plasmid DNA was delivered using ultrasound in the same animal model, leading to transient expression and secretion of BMP-6 localized to the fracture area. Micro-computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to complete radiographic and functional fracture healing in all animals 6 weeks after treatment, whereas nonunion was evident in control animals. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchymal progenitor cells can effectively treat nonhealing bone fractures in large animals, thereby addressing a major orthopedic unmet need and offering new possibilities for clinical translation.
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Células Madre Mesenquimatosas/metabolismo , Células Madre/metabolismo , Ingeniería de Tejidos/métodos , Animales , Proteína Morfogenética Ósea 6/metabolismo , Regeneración Ósea/fisiología , Células Madre Mesenquimatosas/citología , Microburbujas , Células Madre/citología , Porcinos , Porcinos EnanosRESUMEN
BACKGROUND: In hypertensive patients, the polymorphonuclear leukocytes (PMNLs) are primed, concomitantly contributing to oxidative stress and chronic low-grade inflammation. Furthermore, in the Sabra rat model of salt-induced hypertension, priming of PMNLs, oxidative stress and inflammation antecede the development of hypertension. In the present study we tested the hypothesis that PMNL priming and PMNL and white blood cells (WBC) counts are interrelated with blood pressure values. Therefore, we have evaluated the correlation between WBC and PMNL counts, PMNL priming parameters and blood pressure in untreated essential hypertension patients (EH) and age and gender healthy controls. METHODS: Diastolic blood pressure (DBP), systolic blood pressure (SBP) and mean arterial pressure (MAP) values were correlated by linear regression analysis with the rates of superoxide release from separated PMNLs and with WBC and PMNL counts. RESULTS: The rate of superoxide release from PMNLs was higher in EH patients compared to their healthy controls. The rate of superoxide release from PMNLs correlated with SBP, DBP and MAP. WBC and PMNL counts were all significantly correlated with blood pressure values. CONCLUSION: The results of the study offer an additional mechanism involving primed PMNLs and elevated PMNL counts in the pathogenesis of hypertension and its late cardiovascular complications.
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Presión Sanguínea/fisiología , Hipertensión/fisiopatología , Recuento de Leucocitos , Neutrófilos/fisiología , Adulto , Animales , Diástole , Modelos Animales de Enfermedad , Femenino , Humanos , Hipertensión/sangre , Masculino , Persona de Mediana Edad , Ratas , Ratas Endogámicas , Valores de Referencia , Superóxidos/sangre , SístoleRESUMEN
INTRODUCTION: Oxidative stress (OS), inflammation and insulin resistance are among the mechanisms that have been recently implicated in pathogenesis of essential hypertension (EH). Peripheral polymorphonuclear leukocytes (PMNLs) are primed in EH patients, releasing uncontrolled superoxide anion contributing to OS and chronic low-grade inflammation in these patients. PMNL priming correlates with insulin resistance and with PMNL intracellular calcium ([Ca2+]i). Recent studies have attributed additional anti-oxidative characteristics to the anti-hypertensive drug Lercanidipine (Vasodip), a third generation calcium-channel blocker. AIM: To evaluate possible novel effects of two months of Lercanidipine treatment on systemic and PMNL-related inflammation and on insulin resistance in EH patients. METHODS: Fifteen non-smoking EH patients with untreated mild to moderate high blood pressure (BP) and age- and gender-matched healthy controls (HC) were included in the study. Low-grade inflammation was expressed by PMNL counts and apoptosis, by plasma fibrinogen, CRP and albumin (as a negative acute phase reactant) levels. Fasting serum insulin levels served as a marker of insulin resistance. RESULTS: Inflammation parameters and insulin levels were higher in EH compared to HC. PMNL counts, fibrinogen and insulin levels positively correlated with mean arterial blood pressure values. Two months of Lercanidipine treatment showed a significant decrease in BP, PMNL counts and apoptosis, CRP and serum insulin levels and a significant increase in serum albumin levels. CONCLUSION: The authors imply that the low-grade systemic inflammation and insulin resistance detected in EH patients may be attenuated by the use of Lercanidipine, adding new unknown anti-inflammatory properties to this calcium channel blocker.
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Antihipertensivos/uso terapéutico , Bloqueadores de los Canales de Calcio/uso terapéutico , Dihidropiridinas/uso terapéutico , Hipertensión/tratamiento farmacológico , Hipertensión/fisiopatología , Inflamación/tratamiento farmacológico , Adulto , Anciano , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Humanos , Persona de Mediana Edad , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiologíaRESUMEN
More than 1800 gene therapy clinical trials worldwide have targeted a wide range of conditions including cancer, cardiovascular diseases, and monogenic diseases. Biological (i.e. viral), chemical, and physical approaches have been developed to deliver nucleic acids into cells. Although viral vectors offer the greatest efficiency, they also raise major safety concerns including carcinogenesis and immunogenicity. The goal of microbubble-mediated sonoporation is to enhance the uptake of drugs and nucleic acids. Insonation of microbubbles is thought to facilitate two mechanisms for enhanced uptake: first, deflection of the cell membrane inducing endocytotic uptake, and second, microbubble jetting inducing the formation of pores in the cell membrane. We hypothesized that ultrasound could be used to guide local microbubble-enhanced sonoporation of plasmid DNA. With the aim of optimizing delivery efficiency, we used nonlinear ultrasound and bioluminescence imaging to optimize the acoustic pressure, microbubble concentration, treatment duration, DNA dosage, and number of treatments required for in vivo Luciferase gene expression in a mouse thigh muscle model. We found that mice injected with 50µg luciferase plasmid DNA and 5×10(5) microbubbles followed by ultrasound treatment at 1.4MHz, 200kPa, 100-cycle pulse length, and 540 Hz pulse repetition frequency (PRF) for 2min exhibited superior transgene expression compared to all other treatment groups. The bioluminescent signal measured for these mice on Day 4 post-treatment was 100-fold higher (p<0.0001, n=5 or 6) than the signals for controls treated with DNA injection alone, DNA and microbubble injection, or DNA injection and ultrasound treatment. Our results indicate that these conditions result in efficient gene delivery and prolonged gene expression (up to 21days) with no evidence of tissue damage or off-target delivery. We believe that these promising results bear great promise for the development of microbubble-enhanced sonoporation-induced gene therapies.
Asunto(s)
Técnicas de Transferencia de Gen , Microburbujas , Ondas Ultrasónicas , Animales , ADN/administración & dosificación , Femenino , Expresión Génica , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Músculo Esquelético/metabolismo , Plásmidos , PorosidadRESUMEN
: Mesenchymal stem cells (MSCs) are currently the most established cells for skeletal tissue engineering and regeneration; however, their availability and capability of self-renewal are limited. Recent discoveries of somatic cell reprogramming may be used to overcome these challenges. We hypothesized that induced pluripotent stem cells (iPSCs) that were differentiated into MSCs could be used for bone regeneration. Short-term exposure of embryoid bodies to transforming growth factor-ß was used to direct iPSCs toward MSC differentiation. During this process, two types of iPSC-derived MSCs (iMSCs) were identified: early (aiMSCs) and late (tiMSCs) outgrowing cells. The transition of iPSCs toward MSCs was documented using MSC marker flow cytometry. Both types of iMSCs differentiated in vitro in response to osteogenic or adipogenic supplements. The results of quantitative assays showed that both cell types retained their multidifferentiation potential, although aiMSCs demonstrated higher osteogenic potential than tiMSCs and bone marrow-derived MSCs (BM-MSCs). Ectopic injections of BMP6-overexpressing tiMSCs produced no or limited bone formation, whereas similar injections of BMP6-overexpressing aiMSCs resulted in substantial bone formation. Upon orthotopic injection into radial defects, all three cell types regenerated bone and contributed to defect repair. In conclusion, MSCs can be derived from iPSCs and exhibit self-renewal without tumorigenic ability. Compared with BM-MSCs, aiMSCs acquire more of a stem cell phenotype, whereas tiMSCs acquire more of a differentiated osteoblast phenotype, which aids bone regeneration but does not allow the cells to induce ectopic bone formation (even when triggered by bone morphogenetic proteins), unless in an orthotopic site of bone fracture. SIGNIFICANCE: Mesenchymal stem cells (MSCs) are currently the most established cells for skeletal tissue engineering and regeneration of various skeletal conditions; however, availability of autologous MSCs is very limited. This study demonstrates a new method to differentiate human fibroblast-derived induced pluripotent stem cells (iPSCs) to cells with MSC properties, which we comprehensively characterized including differentiation potential and transcriptomic analysis. We showed that these iPS-derived MSCs are able to regenerate nonunion bone defects in mice more efficiently than bone marrow-derived human MSCs when overexpressing BMP6 using a nonviral transfection method.
RESUMEN
BACKGROUND: Intracellular ionized calcium ([Ca2+]i) is a key mediator in the activation and oxidant production by peripheral polymorphonuclear leukocytes (PMN). Primed PMN contribute to oxidative stress (OS) and inflammation in essential hypertension (EH). Elevated [Ca2+]i has been described in insulin-resistant states and in various cell types in EH but not in EH PMN. The aim of this study was to evaluate the levels of [Ca2+]i in peripheral EH PMN in relation to plasma insulin levels and blood pressure (BP). METHODS: The PMN were separated from blood of 20 nonsmoking, nonobese untreated EH patients, age range 20 to 60 years and from 20 age- and gender-matched healthy individuals (NC). Plasma glucose and insulin levels 2 h after a 75-g oral glucose load, reflected insulin resistance. PMN [Ca2+]i was measured by flow cytometry in isolated cells stained with Fluo-3. RESULTS: The EH PMNs showed significantly increased [Ca2+]i compared to NC PMN. Eighty percent of EH patients showed significantly higher plasma insulin levels after glucose load. Linear regression analysis showed significant correlation between 1) PMN [Ca2+]i and mean arterial pressure (MAP) (r = 0.5, P < .006); 2) PMN [Ca2+]i and fasting plasma insulin (r = 0.7, P < .005); and 3) fasting plasma insulin and MAP (r = 0.4, P < .04). CONCLUSIONS: This study adds PMN to previously described cells exhibiting elevated [Ca2+]i, contributing to OS and inflammation. The correlation of individual BP with both PMN [Ca2+]i and plasma insulin levels, together with the fact that elevated [Ca2+]i mediates PMN priming, suggest that elevated [Ca2+]i and insulin are involved in the pathogenesis of hypertension-induced vascular injury in EH.