In vivo implantation of a tissue engineered stem cell seeded hemi-laryngeal replacement maintains airway, phonation, and swallowing in pigs.

Laryngeal functional impairment relating to swallowing, vocalisation, and respiration can be life changing and devastating for patients. A tissue engineering approach to regenerating vocal folds would represent a significant advantage over current clinical practice. Porcine hemi-larynx were de-cellularised under negative pressure. The resultant acellular scaffold was seeded with human bone marrow derived mesenchymal stem cells and primary human epithelial cells. Seeded scaffolds were implanted orthotopically into a defect created in the thyroid cartilage in 8 pigs and monitored in vivo for 2 months. In vivo assessments consisted of mucosal brushing and bronchoscopy at 1, 2, 4, and 8 weeks post implantation followed by histological evaluation post termination. The implanted graft had no adverse effect on respiratory function in 6 of the 8 pigs; none of the pigs had problems with swallowing or vocalisation. Six out of the 8 animals survived to the planned termination date; 2 animals were terminated due to mild stenosis and deep tissue abscess formation, respectively. Human epithelial cells from mucosal brushings could only be identified at Weeks 1 and 4. The explanted tissue showed complete epithelialisation of the mucosal surface and the development of rudimentary vocal folds. However, there was no evidence of cartilage remodelling at the relatively early censor point. Single stage partial laryngeal replacement is a safe surgical procedure. Replacement with a tissue engineered laryngeal graft as a single procedure is surgically feasible and results in appropriate mucosal coverage and rudimentary vocal fold development.

Characterization of a biologically derived rabbit tracheal scaffold.

There is a clinical need to provide replacement tracheal tissue for the pediatric population affected by congenital defects, as current surgical solutions are not universally applicable. A potential solution is to use tissue engineered scaffold as the framework for regenerating autologous tissue. Rabbit trachea were used and different detergents (Triton x-100 and sodium deoxycholate) and enzymes (DNAse/RNAse) investigated to create a decellularization protocol. Each reagent was initially tested individually and the outcome used to design a combined protocol. At each stage the resultant scaffold was assessed histologically, molecularly for acellularity and matrix preservation. Immunogenicity of the final scaffold was assessed by implantation into a rat model for 4 weeks. Both enzymes and detergents were required to produce a completely acellular (DNA content 42.78 ng/mg) scaffold with preserved collagen and elastin however, GAG content were reduced (8.78 ± 1.35 vs. 5.5 ± 4.8). Following in vivo implantation the scaffold elicited minimal immune response and showed significant cellular infiltration and vasculogenesis. The luminal aspect of the implanted scaffold showed infiltration of host derived cells, which were positive for pan cytokeratin. It is possible to create biologically derived biocompatible scaffolds to address specific pediatric clinical problems. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2126-2135, 2017.

Pilot study of a novel vacuum-assisted method for decellularization of tracheae for clinical tissue engineering applications.

Tissue engineered tracheae have been successfully implanted to treat a small number of patients on compassionate grounds. The treatment has not become mainstream due to the time taken to produce the scaffold and the resultant financial costs. We have developed a method for decellularization (DC) based on vacuum technology, which when combined with an enzyme/detergent protocol significantly reduces the time required to create clinically suitable scaffolds. We have applied this technology to prepare porcine tracheal scaffolds and compared the results to scaffolds produced under normal atmospheric pressures. The principal outcome measures were the reduction in time (9 days to prepare the scaffold) followed by a reduction in residual DNA levels (DC no-vac: 137.8±48.82 ng/mg vs. DC vac 36.83±18.45 ng/mg, p<0.05.). Our approach did not impact on the collagen or glycosaminoglycan content or on the biomechanical properties of the scaffolds. We applied the vacuum technology to human tracheae, which, when implanted in vivo showed no significant adverse immunological response. The addition of a vacuum to a conventional decellularization protocol significantly reduces production time, whilst providing a suitable scaffold. This increases clinical utility and lowers production costs. To our knowledge this is the first time that vacuum assisted decellularization has been explored. Copyright © 2015 John Wiley & Sons, Ltd.

Characterisation of porcine dermis scaffolds decellularised using a novel non-enzymatic method for biomedical applications.

Off-the-shelf availability of tissue-engineered skin constructs, tailored by different combinations of reagents to produce a highly preserved biological matrix is often the only means to help patients suffering skin damage. This study assessed the effect of five different decellularisation methods on porcine dermal scaffolds with regard to matrix composition, biomechanical strength, and cytotoxicity using an in vitro biocompatibility assay. Results demonstrated that four out of the five tested decellularisation protocols were efficient in producing acellular scaffolds. Nevertheless, decellularisation method using osmotic shock without enzymatic digestion showed to be efficient not only in removing cellular material and debris from dermal scaffolds but was also beneficial in the preservation of extracellular matrix components (glycosaminoglycans and collagen). Histological assessment revealed that the dermal architecture of coarse collagen bundles was preserved. Examinations by scanning electron microscopy and transmission electron microscopy showed that the arrangement and ultrastructure of collagen fibrils in the scaffolds were retained following non-enzymatic method of decellularisation and also after collagen crosslinking using genipin. Moreover, this decellularised scaffold was not only shown to be biologically compatible when co-cultured with bone marrow-derived mesenchymal stem cells and fibroblasts, but also stimulated the cells to release trophic factors essential for tissue regeneration.

Ultrasound-guided spinal anaesthesia in obstetrics: is there an advantage over the landmark technique in patients with easily palpable spines?

BACKGROUND: Data are scarce on the advantage of ultrasound-guided spinal anaesthesia in patients with easily identifiable bony landmarks. In this study, we compared the use of ultrasound to the landmark method in patients with no anticipated technical difficulty, presenting for caesarean delivery under spinal anaesthesia. METHODS: A total of 150 pregnant women were recruited in this randomized, controlled study. Ultrasound examination and spinal anaesthesia were performed by three anaesthetists with experience in ultrasound-guided neuraxial block. Patients were randomized to either the Ultrasound Group (n=75) or the Landmark Group (n=75). In both groups the level of L3-4 or L4-5 was identified by ultrasound (transverse and longitudinal approach) or palpation. The primary outcome was the procedure time, measured from the time of skin puncture by the introducer to the time of viewing cerebrospinal fluid at the hub of the spinal needle. Secondary outcomes were the number of skin punctures, number of passes, and incidence of successful spinal blockade. RESULTS: The average procedure time, number of skin punctures and needle passes, were similar in both groups. The number of patients with successful spinal anaesthesia after one puncture was not statistically different between the groups. CONCLUSION: The present results indicate that when performed by anaesthetists experienced in both ultrasound and landmark techniques, the use of ultrasound does not appear to increase the success rate of spinal anaesthesia, or reduce the procedure time or number of attempts in obstetric patients with easily palpable spines.

Neocellularization and neovascularization of nanosized bioactive glass-coated decellularized trabecular bone scaffolds.

In this study, the in vivo recellularization and neovascularization of nanosized bioactive glass (n-BG)-coated decellularized trabecular bone scaffolds were studied in a rat model and quantified using stereological analyses. Based on the highest amount of vascular endothelial growth factor (VEGF) secreted by human fibroblasts grown on n-BG coatings (0-1.245 mg/cm(2)), decellularized trabecular bone samples (porosity: 43-81%) were coated with n-BG particles. Grown on n-BG particles at a coating density of 0.263 mg/cm(2), human fibroblasts produced 4.3 times more VEGF than on uncoated controls. After 8 weeks of implantation in Sprague-Dawley rats, both uncoated and n-BG-coated samples were well infiltrated with newly formed tissue (47-48%) and blood vessels (3-4%). No significant differences were found in cellularization and vascularization between uncoated bone scaffolds and n-BG-coated scaffolds. This finding indicates that the decellularized bone itself may exhibit growth-promoting properties induced by the highly interconnected pore microarchitecture and/or proteins left behind on decellularized scaffolds. Even if we did not find proangiogenic effects in n-BG-coated bone scaffolds, a bioactive coating is considered to be beneficial to impart osteoinductive and osteoconductive properties to decellularized bone. n-BG-coated bone grafts have thus high clinical potential for the regeneration of complex tissue defects given their ability for recellularization and neovascularization.

A novel method for visualising and quantifying through-plane skin layer deformations.

Skin is a multilayer composite and exhibits highly non-linear, viscoelastic, anisotropic material properties. In many consumer product and medical applications (e.g. during shaving, needle insertion, patient re-positioning), large tissue displacements and deformations are involved; consequently large local strains in the skin tissue can occur. Here, we present a novel imaging-based method to study skin deformations and the mechanics of interacting skin layers of full-thickness skin. Shear experiments and real-time video recording were combined with digital image correlation and strain field analysis to visualise and quantify skin layer deformations during dynamic mechanical testing. A global shear strain of 10% was applied to airbrush-patterned porcine skin (thickness: 1.2-1.6mm) using a rotational rheometer. The recordings were analysed with ARAMIS image correlation software, and local skin displacement, strain and stiffness profiles through the skin layers determined. The results of this pilot study revealed inhomogeneous skin deformation, characterised by a gradual transition from a low (2.0-5.0%; epidermis) to high (10-22%; dermis) shear strain regime. Shear moduli ranged from 20 to 130kPa. The herein presented method will be used for more extended studies on viable human skin, and is considered a valuable foundation for further development of constitutive models which can be used in advanced finite element analyses of skin.

The relationship of various arch forms and cortical bone thickness.

OBJECTIVE: Implants are being used in orthodontics as a reliable mode of anchorage. Among other factors, the cortical bone thickness plays a major role in determining the stability of these implants. The objective of this study was to study the relationship of various arch forms and the cortical bone thickness and to determine if the cortical bone thickness varies between various arch forms. This would help to determine the ideal length of an implant for a particular arch form. MATERIALS AND METHODS: A cross sectional tomograph was obtained from 30 patients. Based on arch forms the patients' tomographs were equally divided into three basic square, tapered and ovoid categories, each consisting of 10 patients. Consequently, their buccal and lingual cortical plate thicknesses were measured. RESULTS: The results showed that there was a statistically significant difference between the three arch forms, in which the square arch form had the greatest cortical bone thickness among the three arch forms. CONCLUSION: Patients having a tapered arch form may require implants with greater length than patients having a square or an ovoid arch form. Since the availability of the cortical bone in square arch patients is greater, there is more stability for the implants in these cases; therefore, implants with a shorter length may be used in these cases.

Pediatric iron preparations for infants in Bahrain: some therapeutic concerns.

BACKGROUND: Infants and children are at a high risk for medication errors. OBJECTIVES: This retrospective study was conducted to determine the type and prevalence of prescribing errors related to pediatric iron preparations prescribed in primary care in Bahrain. METHODS: Prescriptions issued for infants and collected at 20 health center pharmacies for 2 weeks were audited, specifically for errors. RESULTS: Of 2,282 prescriptions dispensed for infants (mean age 9.14 +/- 0.91 months), 159 (7.0%) included an iron preparation. Iron preparations were mostly prescribed (90.6%) with brand names, several of which were neither listed in the primary care drug list nor were available as pediatric dosage forms. 42 (26.4%) prescriptions were issued without specifying the dosage forms, 14 (8.8%) without the duration of therapy and 4 (2.5%) without dosage. Iron dosage was stated as metric volume (ml) and metric weight (mg elemental iron) units in 78.6% and 9.4% of the prescriptions, respectively. The mean elemental iron (+/- SD) prescribed for treating anemia was 4.5 +/- 1.7 mg/kg body weight. A significant difference was observed between physicians and nurses regarding the amount of elemental iron prescribed for treating anemia. CONCLUSIONS: Prescribing of multiple brands of pediatric iron preparations unavailable in the primary care drug list and in pediatric dosage forms, prescribing iron as inconvenient decimal fractions (metric volume units), and omission errors in prescriptions, were common. This may be related to poor communications between the prescribers and the pharmacy services and a lack of information dissemination on newly introduced iron formulations. Moreover, frequent changes in brand availability in primary care may have created confusion for prescribers. The communication between pharmacy services and prescribers should be strengthened, and the procurement of multiple brands should be discouraged. A better management of drug supply and effective policies to minimize prescribing errors are needed in Bahrain.

Analysis of drug prescriptions in primary health care centres in Bahrain

The aim of this study was to analyse drug prescribing practices in primary health care centres in Bahrain. We retrospectively evaluated 600 prescriptions selected randomly from all primary health care centres in Bahrain [n = 20] in 2004. Analysis followed WHO recommended prescribing core indicators. The mean number of drugs prescribed at each encounter was 3.3 [SD 0.7]. A single drug was prescribed on 6.3% of prescriptions and drugs were prescribed by generic name on 10.2%. The percentage of total prescriptions for antibiotics was 45.8%, for injections was 9.3% and for vitamins was 12.5%. The prescribing pattern in primary health care centres in Bahrain is associated with polypharmacy, over-prescribing of antibiotics and an under-prescribing of drugs by generic names

Medical mortality in Pakistan: experience at a tertiary care hospital.

AIM: To acquire systematic data on the causes of hospital mortality in Pakistan, a developing country with scant mortality records. STUDY DESIGN: Retrospective review of death certificates and hospital charts of patients dying on general and specialty medical services at our hospital during one calendar year. RESULTS: Of a total 10,590 admissions, 657 (6.2%) died in hospital. The deceased included 357 (54.4%) males and 299 (45.6%) females, with a collective median age of 63 years and mean length of stay 6.71 days (median 4 days, range 1-56 days). Primary cause of death was categorised as infectious (21.2%), pulmonary (17.2%), cancer related (15.7%), cardiovascular (12.6%), gastrointestinal and hepatic (10.8%), neurological (11.4%) and miscellaneous (11.1%). Within each category, the most common diagnoses were septicaemia (76.9% of infectious cases), pneumonia (55.7% of pulmonary cases), myocardial infarction (40.9% of cardiovascular), intracranial haemorrhage (37.3% of neurological), and cirrhosis (45.0% of gastrointestinal). There were multiple causes among malignant disorders with no single cause dominating. Patients with cardiovascular and pulmonary deaths tended to be older than the median age (p = 0.001), while patients with gastrointestinal and cancer related deaths tended to be younger than the median age (p = 0.001). Length of stay did not differ significantly among the various subgroups. About a quarter (26.4%) deaths occurred within 24 h of admission. CONCLUSIONS: Infections, including septicaemia and pneumonia, are the leading causes of hospital mortality in our setting, followed by malignancy and cardiovascular causes. The overall mortality rate is comparable to published mortality data from other hospital settings.

Pneumomediastinum, stomach wall and hepatic portal vein gas secondary to partial necrosis of the stomach wall.

The combination of pneumomediastinum, gastric wall gas and hepatic portal vein gas is a challenging clinical problem. Although different causes of the individual gas sign have been reported in the literature, the cause of a triad of these signs in a single patient is less clear, and represents an extremely rare condition. A 65-year-old man presented with severe lower chest and epigastric pain of a few hours' duration. Initial assessment confirmed epigastric tenderness. Computed tomography showed pneumomediastinum, air in the stomach wall, hepatic portal vein gas and bowel dilatation. Small bowel and right colon dilatation was confirmed at laparotomy. The patient was treated subsequently with antibiotics to cover Gram-positive and Gram-negative bacteria, and anaerobes. The patient was discharged in good general condition on the 12th postoperative day. In conclusion, the triad of pneumomediastinum, gastric wall gas and hepatic portal vein gas is an extremely rare condition and associated with gastric necrosis.

Double immuno-labelling of proliferating villous cytotrophoblasts in thick paraffin sections: integrating immuno-histochemistry and stereology in the human placenta.

In order to understand the pathological basis of abnormal villous trophoblast development in diseased placentas, the organ must be sampled by non-biased methods and subject to analysis by stereological tools. This approach permits quantification of cytotrophoblast density and syncytiotrophoblast structure including evidence of apoptotic shedding via syncytial knots. The stereological quantification of cells (or their) nuclei requires that each should be unambiguously identified and counted within a defined volume of tissue. A major limitation of such studies at present is the inability to accurately identify and phenotype subsets of villous cytotrophoblasts that either proliferate or are destined to fuse into the overlying syncytiotrophoblast. We describe the development of a novel double immuno-labelling protocol to selectively identify proliferating villous cytotrophoblast cells in human placental villi using thick (25microm) paraffin sections suitable for stereological quantification. Cytotrophoblast cells were selectively stained using a monoclonal anti-cytokeratin 7 (CK 7) antibody without antigen retrieval, followed by nuclear Ki-67 co-localisation. Both antibodies displayed full depth penetration with sharp, clearly defined staining precipitates and no cross-reactivity. This double immuno-labelling protocol is reproducible, cost effective and time efficient (8h). Use of a variety of antibodies following antigen retrieval will be a significant advancement in the ability to accurately quantify sub-populations of villous cytotrophoblast in normal and pathological placentas.

IFPA meeting 2008 workshops report.

Workshops are an important part of the IFPA annual meeting. At the IFPA meeting 2008 diverse topics were discussed in 12 themed workshops. Topics covered included: immunology of placentation; galectins and trophoblast invasion; signaling in implantation and invasion; markers to identify trophoblast subpopulations; placental pathology; placental toxicology; stereology; placental transport of fatty acids; placental mesenchymal stem cells; comparative placentation; trophoblast and neoplasia; trophoblast differentiation. This report is a summary of the various topics covered.

Birth injuries in caesarian sections: cases of fracture femur and humerus following caesarian section.

Long bone injuries are less common during caesarian section. Sometimes, they remain unnoticed to the operating surgeon but are frequently noted by attending physician or nurses. The aim of this case study is to remind the surgeon that any forceful extraction may result long bone injuries. So, care should be given during and after delivery to rule out injuries.

Prescription writing skills of residents in a family practice residency programme in Bahrain.

PURPOSE OF THE STUDY: To evaluate the prescription writing skill of final year residents in a family practice residency programme (FPRP) in Bahrain, and to compare skill of residents who have graduated from medical schools with problem based learning (PBL) versus traditional (non-PBL) curricula. STUDY DESIGN: Prescriptions issued by the residents were prospectively collected for two consecutive cohorts in May 2004 and May 2005. Prescription errors were classified as errors of omission (minor and major), commission (incorrect information) and integration (drug-drug interactions). RESULTS: In 69.6% of medications with major omission errors, dosage form (39.4%) and length of treatment (18.5%) were not specified. In 24.7% of medications with commission errors, dosing frequency (19.9%) and incorrect strength/dose (2.2%) were the most common errors. Integration errors comprised 5.7% of all prescribing errors. No significant differences were observed between PBL and non-PBL graduates with regard to the total number of prescriptions with errors, drugs per prescription, polypharmacy, and the total number of drugs with errors. The proportion of prescriptions with a potential for drug-drug interactions was comparable between PBL and non-PBL graduates. PBL graduates prescribed medications using brand names at a rate greater than non-PBL, whereas non-PBL graduates prescribed medications on inappropriate "as required" basis, and injections at a rate greater than PBL residents. CONCLUSIONS: Prescription writing skill of the final year residents in an FPRP programme was suboptimal for both PBL and non-PBL graduates. Integration of prescription writing skill and a rational pharmacotherapeutic programme into the FPRP curriculum is recommended.