Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.
22 Related JoVE Articles!
Handwriting Analysis Indicates Spontaneous Dyskinesias in Neuroleptic Naïve Adolescents at High Risk for Psychosis
Institutions: University of Colorado Boulder, NeuroScript LLC, University of California, San Diego.
Growing evidence suggests that movement abnormalities are a core feature of psychosis. One marker of movement abnormality, dyskinesia, is a result of impaired neuromodulation of dopamine in fronto-striatal pathways. The traditional methods for identifying movement abnormalities include observer-based reports and force stability gauges. The drawbacks of these methods are long training times for raters, experimenter bias, large site differences in instrumental apparatus, and suboptimal reliability. Taking these drawbacks into account has guided the development of better standardized and more efficient procedures to examine movement abnormalities through handwriting analysis software and tablet. Individuals at risk for psychosis showed significantly more dysfluent pen movements (a proximal measure for dyskinesia) in a handwriting task. Handwriting kinematics offers a great advance over previous methods of assessing dyskinesia, which could clearly be beneficial for understanding the etiology of psychosis.
Behavior, Issue 81, Schizophrenia, Disorders with Psychotic Features, Psychology, Clinical, Psychopathology, behavioral sciences, Movement abnormalities, Ultra High Risk, psychosis, handwriting, computer tablet, dyskinesia
Reverse Total Shoulder Arthroplasty
Institutions: Case Western Reserve University.
Reverse total shoulder arthroplasty was initially approved for use in rotator cuff arthropathy and well as chronic pseudoparalysis without arthritis in patients who were not appropriate for tendon transfer reconstructions. Traditional surgical options for these patients were limited and functional results were sub-optimal and at times catastrophic. The use of reverse shoulder arthroplasty has been found to effectively restore these patients function and relieve symptoms associated with their disease. The procedure can be done through two approaches, the deltopectoral or the superolateral. Complication rates associated with the use of the prosthesis have ranged from 8-60% with more recent reports trending lower as experienced is gained. Salvage options for a failed reverse shoulder prosthesis are limited and often have significant associated disability. Indications for the use of this prosthesis continue to be evaluated including its use for revision arthroplasty, proximal humeral fracture and tumor. Careful patient selection is essential because of the significant risks associated with the procedure.
Medicine, Issue 53, Reverse, Total, Shoulder, Arthroplasty, Rotator Cuff, Arthropathy, Arthritis, Glenoid, Humerus, Fracture
The C-seal: A Biofragmentable Drain Protecting the Stapled Colorectal Anastomosis from Leakage
Institutions: University Medical Center Groningen.
Colorectal anastomotic leakage (AL) is a serious complication in colorectal surgery leading to high morbidity and mortality rates1
. The incidence of AL varies between 2.5 and 20% 2-5
. Over the years, many strategies aimed at lowering the incidence of anastomotic leakage have been examined6, 7
The cause of AL is probably multifactorial. Etiological factors include insufficient arterial blood supply, tension on the anastomosis, hematoma and/or infection at the anastomotic site, and co-morbid factors of the patient as diabetes and atherosclerosis8
. Furthermore, some anastomoses may be insufficient from the start due to technical failure.
Currently a new device is developed in our institute aimed at protecting the colorectal anastomosis and lowering the incidence of AL. This so called C-seal is a biofragmentable drain, which is stapled to the anastomosis with the circular stapler. It covers the luminal side of the colorectal anastomosis thereby preventing leakage.
The C-seal is a thin-walled tube-like drain, with an approximate diameter of 4 cm and an approximate length of 25 cm (figure 1). It is a tubular device composed of biodegradable polyurethane. Two flaps with adhesive tape are found at one end of the tube. These flaps are used to attach the C-seal to the anvil of the circular stapler, so that after the anastomosis is made the C-seal can be pulled through the anus. The C-seal remains in situ
for at least 10 days. Thereafter it will lose strength and will degrade to be secreted from the body together with the gastrointestinal natural contents.
The C-seal does not prevent the formation of dehiscences. However, it prevents extravasation of faeces into the peritoneal cavity. This means that a gap at the anastomotic site does not lead to leakage.
Currently, a phase II study testing the C-seal in 35 patients undergoing (colo-)rectal resection with stapled anastomosis is recruiting. The C-seal can be used in both open procedures as well as laparoscopic procedures. The C-seal is only applied in stapled anastomoses within 15cm from the anal verge. In the video, application of the C-seal is shown in an open extended sigmoid resection in a patient suffering from diverticular disease with a stenotic colon.
Medicine, Issue 45, Surgery, low anterior resection, colorectal anastomosis, anastomotic leakage, drain, rectal cancer, circular stapler
Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats
Institutions: Georgetown University, University of Turku Medical Faculty.
Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero
and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.
Medicine, Issue 44, mammary gland morphology, terminal end buds, mammary cancer, maternal dietary exposures, pregnancy, prepubertal dietay exposures
Measuring Oral Fatty Acid Thresholds, Fat Perception, Fatty Food Liking, and Papillae Density in Humans
Institutions: Deakin University.
Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty acid receptors housed on taste cells. Previous research has shown that an individual's oral sensitivity to fatty acid, specifically oleic acid (C18:1) is associated with body mass index (BMI), dietary fat consumption, and the ability to identify fat in foods. We have developed a reliable and reproducible method to assess oral chemoreception of fatty acids, using a milk and C18:1 emulsion, together with an ascending forced choice triangle procedure. In parallel, a food matrix has been developed to assess an individual's ability to perceive fat, in addition to a simple method to assess fatty food liking. As an added measure tongue photography is used to assess papillae density, with higher density often being associated with increased taste sensitivity.
Neuroscience, Issue 88, taste, overweight and obesity, dietary fat, fatty acid, diet, fatty food liking, detection threshold
An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings
Institutions: University of KwaZulu-Natal, Durban, South Africa, Jembi Health Systems, University of Amsterdam, Stanford Medical School.
HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load >1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads <1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.
Medicine, Issue 85, Biomedical Technology, HIV-1, HIV Infections, Viremia, Nucleic Acids, genetics, antiretroviral therapy, drug resistance, genotyping, affordable
Use of Interferon-γ Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus
Institutions: China Medical University , University of Arkansas for Medical Sciences , University of Arkansas for Medical Sciences .
A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-γ (IFN-γ) secretion, and growing and characterizing the T-cell clones (Fig. 1
). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1
. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2
). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- γ secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-γ secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- γ ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2
in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105
cells per well typically used in ELISPOT assays1,3
, 1,000 T-cell clone cells in the presence of 1 x 105
autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-γ. This assures successful performance of IFN-γ ELISPOT assay. Similarly, IFN- γ ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- γ ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4
Immunology, Issue 61, Interferon-γ enzyme-linked immunospot assay, T-cell, epitope, human papillomavirus
Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM
Institutions: U.S. Army Engineer Research and Development Center, University of Alabama, U.S. Army Engineer Research and Development Center.
The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram’s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems1-6
. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula
using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.
Bioengineering, Issue 89, Atractosteus spatula, structure-property relation, nanoindentation, scan electron microscopy, X-ray computed tomography, Fourier transform infrared (FTIR) spectroscopy
Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae
Institutions: Rensselaer Polytechnic Institute.
has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae
has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.
Microbiology, Issue 92, Aging, mutations, genome instability, Saccharomyces cerevisiae, fluctuation test, magnetic sorting, mother cell, replicative aging
Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study
Institutions: RWTH Aachen University, Fraunhofer Gesellschaft.
Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.
Bioengineering, Issue 83, design of experiments (DoE), transient protein expression, plant-derived biopharmaceuticals, promoter, 5'UTR, fluorescent reporter protein, model building, incubation conditions, monoclonal antibody
Implantation of the Syncardia Total Artificial Heart
Institutions: Virginia Commonwealth University, Virginia Commonwealth University.
With advances in technology, the use of mechanical circulatory support devices for end stage heart failure has rapidly increased. The vast majority of such patients are generally well served by left ventricular assist devices (LVADs). However, a subset of patients with late stage biventricular failure or other significant anatomic lesions are not adequately treated by isolated left ventricular mechanical support. Examples of concomitant cardiac pathology that may be better treated by resection and TAH replacement includes: post infarction ventricular septal defect, aortic root aneurysm / dissection, cardiac allograft failure, massive ventricular thrombus, refractory malignant arrhythmias (independent of filling pressures), hypertrophic / restrictive cardiomyopathy, and complex congenital heart disease. Patients often present with cardiogenic shock and multi system organ dysfunction. Excision of both ventricles and orthotopic replacement with a total artificial heart (TAH) is an effective, albeit extreme, therapy for rapid restoration of blood flow and resuscitation. Perioperative management is focused on end organ resuscitation and physical rehabilitation. In addition to the usual concerns of infection, bleeding, and thromboembolism common to all mechanically supported patients, TAH patients face unique risks with regard to renal failure and anemia. Supplementation of the abrupt decrease in brain natriuretic peptide following ventriculectomy appears to have protective renal effects. Anemia following TAH implantation can be profound and persistent. Nonetheless, the anemia is generally well tolerated and transfusion are limited to avoid HLA sensitization. Until recently, TAH patients were confined as inpatients tethered to a 500 lb pneumatic console driver. Recent introduction of a backpack sized portable driver (currently under clinical trial) has enabled patients to be discharged home and even return to work. Despite the profound presentation of these sick patients, there is a 79-87% success in bridge to transplantation.
Medicine, Issue 89, mechanical circulatory support, total artificial heart, biventricular failure, operative techniques
Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction
Institutions: Rutgers University, Koç University, New York University, Fairfield University.
We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the "cloud" and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in cabinet; two such cabinets (16 environments) may be controlled by one computer.
Behavior, Issue 84, genetics, cognitive mechanisms, behavioral screening, learning, memory, timing
Substernal Thyroid Biopsy Using Endobronchial Ultrasound-guided Transbronchial Needle Aspiration
Institutions: State University of New York, Buffalo, Roswell Park Cancer Institute, State University of New York, Buffalo.
Substernal thyroid goiter (STG) represents about 5.8% of all mediastinal lesions1
. There is a wide variation in the published incidence rates due to the lack of a standardized definition for STG. Biopsy is often required to differentiate benign from malignant lesions. Unlike cervical thyroid, the overlying sternum precludes ultrasound-guided percutaneous fine needle aspiration of STG. Consequently, surgical mediastinoscopy is performed in the majority of cases, causing significant procedure related morbidity and cost to healthcare. Endobronchial Ultrasound-guided Transbronchial Needle Aspiration (EBUS-TBNA) is a frequently used procedure for diagnosis and staging of non-small cell lung cancer (NSCLC). Minimally invasive needle biopsy for lesions adjacent to the airways can be performed under real-time ultrasound guidance using EBUS. Its safety and efficacy is well established with over 90% sensitivity and specificity. The ability to perform EBUS as an outpatient procedure with same-day discharges offers distinct morbidity and financial advantages over surgery. As physicians performing EBUS gained procedural expertise, they have attempted to diversify its role in the diagnosis of non-lymph node thoracic pathologies. We propose here a role for EBUS-TBNA in the diagnosis of substernal thyroid lesions, along with a step-by-step protocol for the procedure.
Medicine, Issue 93, substernal thyroid, retrosternal thyroid, intra-thoracic thyroid, goiter, endobronchial ultrasound, EBUS, transbronchial needle aspiration, TBNA, biopsy, needle biopsy
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro
. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro
replication of HIV-1 as influenced by the gag
gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag
gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro
replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects
Institutions: University of California, San Francisco .
Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens4
. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice 5-7
, after application of bioactive molecules 8,9
, after altering physiological parameters of healing 10
, after modifying the amount or time of stabilization 11
, after distraction osteogenesis 12
, after creation of a non-union 13
, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.
Medicine, Issue 62, Bone fracture, intramembranous ossification, distraction osteogenesis, bone healing
Adjustable Stiffness, External Fixator for the Rat Femur Osteotomy and Segmental Bone Defect Models
Institutions: Queensland University of Technology, RISystem AG.
The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there has been great clinical interest in improving bone healing by altering the mechanical environment through the fixation stability around the lesion. One constraint of preclinical animal research in this area is the lack of experimental control over the local mechanical environment within a large segmental defect as well as osteotomies as they heal. In this paper we report on the design and use of an external fixator to study the healing of large segmental bone defects or osteotomies. This device not only allows for controlled axial stiffness on the bone lesion as it heals, but it also enables the change of stiffness during the healing process in vivo.
The conducted experiments have shown that the fixators were able to maintain a 5 mm femoral defect gap in rats in vivo
during unrestricted cage activity for at least 8 weeks. Likewise, we observed no distortion or infections, including pin infections during the entire healing period. These results demonstrate that our newly developed external fixator was able to achieve reproducible and standardized stabilization, and the alteration of the mechanical environment of in vivo
rat large bone defects and various size osteotomies. This confirms that the external fixation device is well suited for preclinical research investigations using a rat model in the field of bone regeneration and repair.
Medicine, Issue 92, external fixator, bone healing, small animal model, large bone defect and osteotomy model, rat model, mechanical environment, mechanobiology.
Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy
Institutions: The University of North Carolina at Chapel Hill.
Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum “cast” intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.
Biophysics, Issue 91, Freeze-fracture; Freeze-etch; Membranes; Intercellular junctions; Materials science; Nanotechnology; Electron microscopy
Community-based Adapted Tango Dancing for Individuals with Parkinson's Disease and Older Adults
Institutions: Emory University School of Medicine, Brigham and Woman‘s Hospital and Massachusetts General Hospital.
Adapted tango dancing improves mobility and balance in older adults and additional populations with balance impairments. It is composed of very simple step elements. Adapted tango involves movement initiation and cessation, multi-directional perturbations, varied speeds and rhythms. Focus on foot placement, whole body coordination, and attention to partner, path of movement, and aesthetics likely underlie adapted tango’s demonstrated efficacy for improving mobility and balance. In this paper, we describe the methodology to disseminate the adapted tango teaching methods to dance instructor trainees and to implement the adapted tango by the trainees in the community for older adults and individuals with Parkinson’s Disease (PD). Efficacy in improving mobility (measured with the Timed Up and Go, Tandem stance, Berg Balance Scale, Gait Speed and 30 sec chair stand), safety and fidelity of the program is maximized through targeted instructor and volunteer training and a structured detailed syllabus outlining class practices and progression.
Behavior, Issue 94, Dance, tango, balance, pedagogy, dissemination, exercise, older adults, Parkinson's Disease, mobility impairments, falls
Laparoscopic Left Liver Sectoriectomy of Caroli's Disease Limited to Segment II and III
Institutions: University of Insubria, University of Insubria.
Caroli's disease is defined as a abnormal dilatation of the intra-hepatica bile ducts: Its incidence is extremely low (1 in 1,000,000 population) and in most of the cases the whole liver is interested and liver transplantation is the treatment of choice. In case of dilatation limited to the left or right lobe, liver resection can be performed. For many year the standard approach for liver resection has been a formal laparotomy by means of a large incision of abdomen that is characterized by significant post-operatie morbidity. More recently, minimally invasive, laparoscopic approach has been proposed as possible surgical technique for liver resection both for benign and malignant diseases. The main benefits of the minimally invasive approach is represented by a significant reduction of the surgical trauma that allows a faster recovery a less post-operative complications.
This video shows a case of Caroli s disease occured in a 58 years old male admitted at the gastroenterology department for sudden onset of abdominal pain associated with fever (>38C° ), nausea and shivering. Abdominal ultrasound demonstrated a significant dilatation of intra-hepatic left sited bile ducts with no evidences of gallbladder or common bile duct stones. Such findings were confirmed abdominal high resolution computer tomography.
Laparoscopic left sectoriectomy was planned. Five trocars and 30° optic was used, exploration of the abdominal cavity showed no adhesions or evidences of other diseases.
In order to control blood inflow to the liver, vascular clamp was placed on the hepatic pedicle (Pringle s manouvre), Parenchymal division is carried out with a combined use of 5 mm bipolar forceps and 5 mm ultrasonic dissector. A severely dilated left hepatic duct was isolated and divided using a 45mm endoscopic vascular stapler. Liver dissection was continued up to isolation of the main left portal branch that was then divided with a further cartridge of 45 mm vascular stapler.
At his point the left liver remains attached only by the left hepatic vein: division of the triangular ligament was performed using monopolar hook and the hepatic vein isolated and the divided using vascular stapler.
Haemostatis was refined by application of argon beam coagulation and no bleeding was revealed even after removal of the vascular clamp (total Pringle s time 27 minutes).
Postoperative course was uneventful, minimal elevation of the liver function tests was recorded in post-operative day 1 but returned to normal at discharged on post-operative day 3.
Medicine, Issue 24, Laparoscopy, Liver resection, Caroli's disease, Left sectoriectomy
Right Hemihepatectomy by Suprahilar Intrahepatic Transection of the Right Hemipedicle using a Vascular Stapler
Institutions: Tübingen University Hospital.
Successful hepatic resection requires profound anatomical knowledge and delicate surgical technique. Hemihepatectomies are mostly performed after preparing the extrahepatic hilar structures within the hepatoduodenal ligament, even in benign tumours or liver metastasis.1-5
. Regional extrahepatic lymphadenectomy is an oncological standard in hilar cholangiocarcinoma, intrahepatic cholangio-cellular carcinoma and hepatocellular carcinoma, whereas lymph node metastases in the hepatic hilus in patients with liver metastasis are rarely occult. Major disadvantages of these procedures are the complex preparation of the hilus with the risk of injuring contralateral structures and the possibility of bleeding from portal vein side-branches or impaired perfusion of bile ducts. We developed a technique of right hemihepatectomy or resection of the left lateral segments with intrahepatic transection of the pedicle that leaves the hepatoduodenal ligament completely untouched. 6
However, if intraoperative visualization or palpation of the ligament is suspicious for tumor infiltration or lymph node metastasis, the hilus should be explored and a lymphadenectomy performed.
Medicine, Issue 35, Liver resection, liver tumour, intrahepatic hilus stapling, right hemipedicle
A New Single Chamber Implantable Defibrillator with Atrial Sensing: A Practical Demonstration of Sensing and Ease of Implantation
Institutions: University Hospital of Rostock, Germany.
Implantable cardioverter-defibrillators (ICDs) terminate ventricular tachycardia (VT) and ventricular fibrillation (VF) with high efficacy and can protect patients from sudden cardiac death (SCD). However, inappropriate shocks may occur if tachycardias are misdiagnosed. Inappropriate shocks are harmful and impair patient quality of life. The risk of inappropriate therapy increases with lower detection rates programmed in the ICD. Single-chamber detection poses greater risks for misdiagnosis when compared with dual-chamber devices that have the benefit of additional atrial information. However, using a dual-chamber device merely for the sake of detection is generally not accepted, since the risks associated with the second electrode may outweigh the benefits of detection. Therefore, BIOTRONIK developed a ventricular lead called the LinoxSMART
S DX, which allows for the detection of atrial signals from two electrodes positioned at the atrial part of the ventricular electrode. This device contains two ring electrodes; one that contacts the atrial wall at the junction of the superior vena cava (SVC) and one positioned at the free floating part of the electrode in the atrium. The excellent signal quality can only be achieved by a special filter setting in the ICD (Lumax 540 and 740 VR-T DX, BIOTRONIK). Here, the ease of implantation of the system will be demonstrated.
Medicine, Issue 60, Implantable defibrillator, dual chamber, single chamber, tachycardia detection
A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia
Institutions: Universite de Montreal, Universite de Montreal, Universite de Montreal.
There are several lines of evidence supporting the role of de novo
mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo
mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo
mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1
and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo
mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2
. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo
mutations. This is the case for autism and schizophrenia3
. The male-to-female ratio of mutation rate is estimated at about 4–6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo
mutations would more frequently come from males, particularly older males4
. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo
mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo
mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.
Medicine, Issue 52, de novo mutation, complex diseases, schizophrenia, autism, rare variations, DNA sequencing