Aquaporin expression in the human and canine intervertebral disc during maturation and degeneration

The intervertebral disc (IVD) is a highly hydrated tissue, the rich proteoglycan matrix imbibes water, enabling the disc to withstand compressive loads. During ageing and degeneration increased matrix degradation leads to dehydration and loss of function. Aquaporins (AQP) are a family of transmembrane channel proteins that selectively allow the passage of water in and out of cells and are responsible for maintaining water homeostasis in many tissues. Here, the expression of all 13 AQPs at gene and protein level was investigated in human and canine non‐degenerate and degenerate IVDs to develop an understanding of the role of AQPs during degeneration. Furthermore, in order to explore the transition of notochordal cells (NCs) towards nucleus pulposus (NP) cells, AQP expression was investigated in canine IVDs enriched in NCs to understand the role of AQPs in IVD maturation. AQP0, 1, 2, 3, 4, 5, 6, 7 and 9 were expressed at gene and protein level in both non‐degenerate and degenerate human NP tissue. AQP2 and 7 immunopositivity increased with degeneration in human NP tissue, whereas AQP4 expression decreased with degeneration in a similar way to AQP 1 and 5 shown previously. All AQP proteins that were identified in human NP tissue were also expressed in canine NP tissue. AQP2, 5, 6 and 9 were found to localise to vacuole‐like membranes and cell membranes in NC cells. In conclusion, AQPs were abundantly expressed in human and canine IVDs. The expression of many AQP isotypes potentially alludes to multi‐faceted functions related to adaption of NP cells to the conditions they encounter within their microenvironment in health and degeneration. The presence of AQPs within the IVD may suggest an adaptive role for these water channels during the development and maintenance of the healthy, mature IVD

The severity of pandemic H1N1 influenza in the United States, from April to July 2009: A Bayesian analysis

Background: Accurate measures of the severity of pandemic (H1N1) 2009 influenza (pH1N1) are needed to assess the likely impact of an anticipated resurgence in the autumn in the Northern Hemisphere. Severity has been difficult to measure because jurisdictions with large numbers of deaths and other severe outcomes have had too many cases to assess the total number with confidence. Also, detection of severe cases may be more likely, resulting in overestimation of the severity of an average case. We sought to estimate the probabilities that symptomatic infection would lead to hospitalization, ICU admission, and death by combining data from multiple sources. Methods and Findings: We used complementary data from two US cities: Milwaukee attempted to identify cases of medically attended infection whether or not they required hospitalization, while New York City focused on the identification of hospitalizations, intensive care admission or mechanical ventilation (hereafter, ICU), and deaths. New York data were used to estimate numerators for ICU and death, and two sources of data - medically attended cases in Milwaukee or self-reported influenza-like illness (ILI) in New York - were used to estimate ratios of symptomatic cases to hospitalizations. Combining these data with estimates of the fraction detected for each level of severity, we estimated the proportion of symptomatic patients who died (symptomatic case-fatality ratio, sCFR), required ICU (sCIR), and required hospitalization (sCHR), overall and by age category. Evidence, prior information, and associated uncertainty were analyzed in a Bayesian evidence synthesis framework. Using medically attended cases and estimates of the proportion of symptomatic cases medically attended, we estimated an sCFR of 0.048% (95% credible interval [CI] 0.026%-0.096%), sCIR of 0.239% (0.134%-0.458%), and sCHR of 1.44% (0.83%-2.64%). Using self-reported ILI, we obtained estimates approximately 7-96lower. sCFR and sCIR appear to be highest in persons aged 18 y and older, and lowest in children aged 5-17 y. sCHR appears to be lowest in persons aged 5-17; our data were too sparse to allow us to determine the group in which it was the highest. Conclusions: These estimates suggest that an autumn-winter pandemic wave of pH1N1 with comparable severity per case could lead to a number of deaths in the range from considerably below that associated with seasonal influenza to slightly higher, but with the greatest impact in children aged 0-4 and adults 18-64. These estimates of impact depend on assumptions about total incidence of infection and would be larger if incidence of symptomatic infection were higher or shifted toward adults, if viral virulence increased, or if suboptimal treatment resulted from stress on the health care system; numbers would decrease if the total proportion of the population symptomatically infected were lower than assumed.published_or_final_versio

Harmonization and standardization of nucleus pulposus cell extraction and culture methods

Background In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab-to-lab variability jeopardizes the much-needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources. Methods The most commonly applied methods for NP cell extraction, expansion, and re-differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re-differentiation media and techniques were also investigated. Results Recommended protocols are provided for extraction, expansion, and re-differentiation of NP cells from common species utilized for NP cell culture. Conclusions This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species-specific pronase usage, 60–100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross-lab comparisons on NP cells worldwide

Harmonization and standardization of nucleus pulposus cell extraction and culture methods

Background: In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab‐to‐lab variability jeopardizes the much‐needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources. Methods: The most commonly applied methods for NP cell extraction, expansion, and re‐differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re‐differentiation media and techniques were also investigated. Results: Recommended protocols are provided for extraction, expansion, and re‐differentiation of NP cells from common species utilized for NP cell culture. Conclusions: This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species‐specific pronase usage, 60–100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross‐lab comparisons on NP cells worldwide

Recommendations for intervertebral disc notochordal cell investigation: From isolation to characterization

International audienceBackground: Lineage-tracing experiments have established that the central region of the mature intervertebral disc, the nucleus pulposus (NP), develops from the embryonic structure called "the notochord". However, changes in the cells derived from the notochord which form the NP (i.e., notochordal cells [NCs]), in terms of their phenotype and functional identity from early developmental stages to skeletal maturation are less understood. These key issues require further investigation to better comprehend the role of NCs in homeostasis and degeneration as well as their potential for regeneration. Progress in utilizing NCs is currently hampered due to poor consistency and lack of consensus methodology for in vitro NC extraction, manipulation, and characterization.Methods: Here, an international group has come together to provide key recommendations and methodologies for NC isolation within key species, numeration, in vitro manipulation and culture, and characterization.Results: Recommeded protocols are provided for isolation and culture of NCs. Experimental testing provided recommended methodology for numeration of NCs. The issues of cryopreservation are demonstrated, and a pannel of immunohistochemical markers are provided to inform NC characterization.Conclusions: Together we hope this article provides a road map for in vitro studies of NCs to support advances in research into NC physiology and their potential in regenerative therapies

Pulpal response of dogs primary teeth to an adhesive system or to a calcium hydroxide cement Resposta pulpar de dentes decíduos de cães a um sistema adesivo ou ao cimento de hidróxido de cálcio

The aim of this study was to evaluate the pulpal response of dogs primary teeth to an adhesive system or to a calcium hidroxide cement after mechanic exposure of the pulp. Three mongrel dogs were used and ten class V cavities were prepared on their teeth. A mechanic pulp exposure was produced with a sterile exploratory probe in the central portion of each cavity and bleeding was controlled with dry sterile cotton pellets. Enamel, dentin and the site of the pulp exposure of five teeth were etched with 35% phosphoric acid followed by the application of an adhesive system (Scotchbond Multi-Purpose - 3M). In the other five teeth, calcium hydroxide cement (Hydro C - Dentsply) was applied on the site of the pulp exposition before application of the adhesive system (Scotchbond Multi-Purpose - 3M). All teeth were restored with a resin composite (Z-100 - 3M). After 7, 30 or 45 days the dogs were anesthetized and perfused with saline followed by a solution of neutral buffered formalin. Maxilla and mandible were sectioned into three parts and placed in a solution for demineralization. Following bone demineralization, all teeth were cut, trimmed, embedded in paraffin and longitudinally cut. Then, the teeth were stained with hematoxilin and eosin and observed under a light microscope. The results obtained with the treatments proposed in this study showed the presence and persistence of an inflammatory response of different intensities at the three experimental periods. There was no variation in the inflammatory response regarding the different treatments performed.<br>O objetivo deste estudo foi de avaliar a resposta pulpar de dentes decíduos de cães à um sistema adesivo ou a um cimento de hidróxido de cálcio após exposição mecânica da polpa. Foram utilizados três cães sem raça definida, e nestes foram realizados dez preparos cavitários classe V. Uma exposição pulpar mecânica foi produzida com uma sonda exploradora esterilizada, na porção central de cada cavidade. A hemorragia foi controlada com "bolinhas" de algodão esterilizadas. O esmalte, dentina e local da exposição pulpar de cinco dentes foram condicionados com ácido fosfórico a 35%, seguido da aplicação de um sistema adesivo (Scotchbond Multi-Uso - 3M). Nos outros cinco dentes, um cimento de hidróxido de cálcio (Hydro C - Dentsply) foi aplicada no local da exposição pulpar antes da aplicação do mesmo sistema adesivo. Todos os dentes foram restaurados com uma resina composta (Z-100 - 3M). Após 7, 30 ou 45 dias os cães foram anestesiados e perfundidos com solução salina seguida de uma solução de formalina neutra tamponada. A maxila e a mandíbula foram seccionadas em três partes e estocadas em uma solução para desmineralização. Após a desmineralização óssea, todos os dentes foram cortados, incluídos em parafina e seccionados longitudinalmente. A seguir todos os dentes foram corados com hematoxilina e eosina e observados em microscópio de luz. Os resultados demonstraram a presença e persistência de uma resposta inflamatória de diferentes intensidades nos três períodos experimentais. Não houve variação na resposta inflamatória pelos diferentes tratamentos propostos