Hyperosmolarity normalises serum-induced changes to chondrocyte properties in a model of cartilage injury.

Partial-thickness cartilage injuries do not heal effectively, potentially leading to degeneration as occurs in post-traumatic osteoarthritis (PTOA). The role of chondrocytes could be crucial in determining the nature of the repair; however, their response to this injury is poorly understood. We have utilised an in vitro bovine osteochondral partial-thickness scalpel injury model and determined chondrocyte properties at and distant from the injury in the presence/absence of (a) serum-free DMEM (340 mOsm), (b) synovial fluid DMEM (SF-DMEM), (c) foetal calf serum DMEM (FCS-DMEM), (d) hyperosmolar serum-free DMEM (600 mOsm), or (e) hyperosmolar FCS-DMEM for up to two weeks. Chondrocytes were fluorescently-labelled with 5-chloromethylfluorescein-diacetate (CMFDA)/propidium iodide (PI) for live/dead cells and imaged using confocal microscopy. Quantitative data were obtained on chondrocyte properties (cell volume, clusters, morphology) at and distant from the injury. In serum-free DMEM, chondrocyte morphology at the injury remained unaffected throughout culture. However, with SF-DMEM or FCS-DMEM the chondrocytes displayed an increase in volume (p < 0.0001), cluster formation (FCS; p < 0.01) and abnormal morphology (p < 0.001) compared to serum-free DMEM. Cluster formation and shape changes during FCS-DMEM culture were more pronounced than with SF-DMEM. SF-DMEM or FCS-DMEM stimulated these changes to chondrocytes at the injury with only small effects on distant cells. Hyperosmolarity inhibited the morphological and volume changes to chondrocytes induced by FCS-DMEM (p < 0.001) and the injured cartilage had the appearance of that in serum-free DMEM. Raised osmolarity may therefore have benefit in preserving the morphological phenotype of chondrocytes at the site of injury, and thus promote more effective integrative repair in partial-thickness cartilage injury.

Airflow accelerates bovine and human articular cartilage drying and chondrocyte death.

OBJECTIVE: Exposure of articular cartilage to static air results in changes to the extracellular matrix (ECM) and stimulates chondrocyte death, which may cause joint degeneration. However during open orthopaedic surgery, cartilage is often exposed to laminar airflow, which may exacerbate these damaging effects. We compared drying in static and moving air in terms of cartilage appearance, hydration and chondrocyte viability, and tested the ability of saline-saturated gauze to limit the detrimental effects of air exposure. DESIGN: Articular cartilage from bovine metatarsophalangeal joints (N = 50) and human femoral heads (N = 6) was exposed for 90 min to (1) static air (2) airflow (up to 0.34 m/s), or (3) airflow (0.18 m/s), covered with gauze. Following air exposure, cartilage was also rehydrated (0.9% saline; 120 min) to determine the reversibility of drying effects. The influence of airflow was assessed by studying macroscopic appearance, and quantifying superficial zone (SZ) chondrocyte viability and cartilage hydration. RESULTS: Airflow caused advanced changes to cartilage appearance, accelerated chondrocyte death, and increased dehydration compared to static air. These effects were prevented if cartilage was covered by saline-saturated gauze. Cartilage rehydration reversed macroscopic changes associated with drying but the chondrocyte death was not altered. Chondrocytes at the cut edge of cartilage were more sensitive to drying compared to cells distant from the edge. CONCLUSIONS: Airflow significantly increased articular cartilage dehydration and chondrocyte death compared to static air. As laminar airflow is routinely utilised in operating theatres, it is essential that articular cartilage is kept wet via irrigation or by covering with saline-saturated gauze to prevent chondrocyte death.

The use of hyperosmotic saline for chondroprotection: implications for orthopaedic surgery and cartilage repair.

OBJECTIVE: Articular cartilage may experience iatrogenic injury during routine orthopaedic/arthroscopic procedures. This could cause chondrocyte death, leading to cartilage degeneration and posttraumatic osteoarthritis. In an in vitro cartilage injury model, chondrocyte death was reduced by increasing the osmolarity of normal saline (NS), the most commonly-used irrigation solution. Here, we studied the effect of hyperosmolar saline (HS) on chondrocyte viability and cartilage repair in an in vivo injury model. DESIGN: Cartilage injury was induced by a single scalpel cut along the patellar groove of 8 week old rats in the absence of irrigation or with either NS (300 mOsm) or HS (600 mOsm). The percentage of cell death (PCD) within the injured area was assessed using confocal microscopy. Repair from injury was evaluated by histology/immunostaining, and inflammatory response by histology, cytokine array analysis and ELISA (enzyme-linked immunosorbent assay). RESULTS: The PCD in saline-irrigated joints was increased compared to non-irrigated (NI) joints [PCD = 20.8% (95%CI; 14.5, 27.1); PCD = 9.14% (95%CI; 6.3, 11.9); P = 0.0017]. However, hyperosmotic saline reduced chondrocyte death compared to NS (PCD = 10.4% (95%CI; 8.5, 12.3) P = 0.0024). Repair score, type II collagen and aggrecan levels, and injury width, were significantly improved with hyperosmotic compared to NS. Mild synovitis and similar changes in serum cytokine profile occurred in all operated joints irrespective of experimental group. CONCLUSIONS: Hyperosmotic saline significantly reduced the chondrocyte death associated with scalpel-induced injury and enhanced cartilage repair. This irrigation solution might be useful as a simple chondroprotective strategy and may also reduce unintentional cartilage injury during articular reconstructive surgery and promote integrative cartilage repair, thereby reducing the risk of posttraumatic osteoarthritis.

TEMS: results of a specialist centre.

INTRODUCTION: Transanal endoscopic microsurgery (TEMS) is becoming more widespread due to the increasing body of evidence to support its role. Previous published data has reported recurrence rates in excess of 10% for benign polyps after TEMS. METHODS: Bradford Royal Infirmary is a tertiary referral centre for TEMS and early rectal cancer in the UK. Data for all TEMS operations were entered into a prospective database over a 7-year period. Demographic data, complications and recurrence rates were recorded. Both benign adenomas and malignant lesions were included. RESULTS: A total of 164 patients (65% male), with a mean age of 68 years were included; 114 (70%) of the lesions resected were benign adenomas, and 50 (30%) were malignant lesions. Median polyp size was 4 (range 0.6-14.5) cm. Mean length of operation was 55 (range 10-120) min. There were no recurrences in any patients with a benign adenoma resected; two patients with malignant lesions developed recurrences. Three intra-operative complications were recorded, two rectal perforations (repaired primarily, one requiring defunctioning stoma), and a further patient suffered a blood loss of >300 ml requiring transfusion. Six patients developed strictures requiring dilation either endoscopically or under anaesthetic in the post-operative period. CONCLUSIONS: We have demonstrated that TEMS procedures performed in a specialist centre provide low rates of both recurrence and complication. Within a specialist centre, TEMS surgery should be offered to all patients for rectal lesions, both benign and malignant, that are amenable to TEMS.

Chondrocyte death after drilling and articular screw insertion in a bovine model.

OBJECTIVE: Intra-articular screws are used for internal fixation of osteochondral fragments after fracture or osteochondritis dissecans. This causes cartilage injury potentially leading to chondrocyte death. We have visualised/quantified the hole and zone of cell death (ZCD) in cartilage after drilling/insertion of various articular screws. METHOD: Using an ex vivo bovine model with transmitted light and confocal laser scanning microscopy (CLSM), the holes and ZCD following drilling/insertion of articular screws (cortical screw, headless variable pitch metallic screw, headless variable pitch bioabsorbable screw) were evaluated. In situ chondrocyte death was determined by live/dead cell viability assay. An imaging/quantification protocol was developed to compare hole diameter and ZCD from drilling/insertion of screws into cartilage. The effect of saline irrigation during drilling on the ZCD was also quantified. RESULTS: Screw insertion created holes in cartilage that were significantly (P ≤ 0.001) less than the diameters of the equipment used. With a 1.5 mm drill, a ZCD of 580.2 ± 124 µm was produced which increased to 637.0 ± 44 µm following insertion of a 2 mm cortical screw although this was not significant (P > 0.05). The ZCD from insertion of the variable pitch headless screws (diam. 3.5 mm) was lower for the metallic compared to the bioabsorbable design (800.9 ± 159 vs 1,236.4 ± 212 µm, respectively; P < 0.01). The ZCD from drilling was reduced ∼50% (P < 0.001) by saline irrigation. CONCLUSIONS: Cartilage injury during intra-articular screw fixation caused a ZCD around the hole irrespective of screw design. Saline irrigation significantly reduced the ZCD from drilling into cartilage.

Rapid in situ chondrocyte death induced by Staphylococcus aureus toxins in a bovine cartilage explant model of septic arthritis.

OBJECTIVE: To assess in situ chondrocyte viability following exposure to a laboratory strain and clinical isolates of Staphylococcus aureus. METHODS: Bovine cartilage explants were cultured in the presence of S. aureus 8325-4 (laboratory strain), clinical S. aureus isolates or non-infected culture medium of pH values 7.4, 6.4 and 5.4. All clinical isolates were isolated from the joint aspirates of patients presenting with S. aureus-induced septic arthritis (SA). At designated time points, in situ chondrocyte viability was assessed within defined regions-of-interest in the axial and coronal plane following live- and dead-cell image acquisition using the fluorescent probes 5-chloromethylfluorescein diacetate (CMFDA) and propidium iodide (PI), respectively, and confocal laser-scanning microscopy (CLSM). Cartilage water content, following S. aureus 8325-4 exposure, was obtained by measuring cartilage wet and dry weights. RESULTS: S. aureus 8325-4 and clinical S. aureus isolates rapidly reduced in situ chondrocyte viability (>45% chondrocyte death at 40 h). The increased acidity, observed during bacterial culture, had a minimal effect on chondrocyte viability. Chondrocyte death commenced within the superficial zone (SZ) and rapidly progressed to the deep zone (DZ). Simultaneous exposure of SZ and DZ chondrocytes to S. aureus 8325-4 toxins found SZ chondrocytes to be more susceptible to the toxins than DZ chondrocytes. Cartilage water content was not significantly altered compared to non-infected controls. CONCLUSIONS: Toxins released by S. aureus have a rapid and fatal action on in situ chondrocytes in this experimental model of SA. These data advocate the prompt and thorough removal of bacteria and their toxins during the treatment of SA.

Ascophyllum nodosum enriched bread reduces subsequent energy intake with no effect on post-prandial glucose and cholesterol in healthy, overweight males. A pilot study.

It is well recognised that the consumption of seaweed isolates (such as alginate) successfully reduce energy intake and modulate glycaemic and cholesterolaemic responses. However, to date, the effect of adding whole seaweed to bread has not been widely investigated. Hence, this study aims to investigate the acceptability of Ascophyllum nodosum enriched bread as part of a meal, and measure its effect on energy intake and nutrient absorption in overweight, healthy males to see if it has a similar impact. Results from the acceptability study, (79 untrained sensory panellists) indicated that it is acceptable to incorporate seaweed (A. nodosum) into a staple food such as bread at concentrations of up to 4% per 400 g wholemeal loaf. A single blind cross over trial (n=12 males, aged 40.1±12.5 years; BMI 30.8±4.4 kg/m(2)) was used to compare energy intake and nutrient uptake after a breakfast meal using the enriched bread (4% A. nodosum) against the control bread (0% A. nodosum). Consumption of the enriched bread at breakfast led to a significant reduction (16.4%) in energy intake at a test meal 4 h later. Differences between treatment arms for area under the curve, peak values, and time of peak for blood glucose and cholesterol were not significant. Further investigation of potential mechanisms of action is warranted.

A cell shrinkage artefact in growth plate chondrocytes with common fixative solutions: importance of fixative osmolarity for maintaining morphology.

The remarkable increase in chondrocyte volume is a major determinant in the longitudinal growth of mammalian bones. To permit a detailed morphological study of hypertrophic chondrocytes using standard histological techniques, the preservation of normal chondrocyte morphology is essential. We noticed that during fixation of growth plates with conventional fixative solutions, there was a marked morphological (shrinkage) artifact, and we postulated that this arose from the hyper-osmotic nature of these solutions. To test this, we fixed proximal tibia growth plates of 7-day-old rat bones in either (a) paraformaldehyde (PFA; 4%), (b) glutaraldehyde (GA; 2%) with PFA (2%) with ruthenium hexamine trichloride (RHT; 0.7%), (c) GA (2%) with RHT (0.7%), or (d) GA (1.3%) with RHT (0.5%) and osmolarity adjusted to a 'physiological' level of approximately 280mOsm. Using conventional histological methods, confocal microscopy, and image analysis on fluorescently-labelled fixed and living chondrocytes, we then quantified the extent of cell shrinkage and volume change. Our data showed that the high osmolarity of conventional fixatives caused a shrinkage artefact to chondrocytes. This was particularly evident when whole bones were fixed, but could be markedly reduced if bones were sagittally bisected prior to fixation. The shrinkage artefact could be avoided by adjusting the osmolarity of the fixatives to the osmotic pressure of normal extracellular fluids ( approximately 280mOsm). These results emphasize the importance of fixative osmolarity, in order to accurately preserve the normal volume/morphology of cells within tissues.

Oligodendrocyte population dynamics and the role of PDGF in vivo.

Oligodendrocyte progenitors originate near the floor plate of the spinal cord, then proliferate and migrate throughout the cord before giving rise to oligodendrocytes. Progenitor cell proliferation stops before birth because the cell cycle slows down, linked to an increase in differentiation and death. Experiments with transgenic mice show that platelet-derived growth factor (PDGF) drives progenitor cell division and suggest that slowing of and exit from the cycle reflects a decline in PDGF signaling. Overexpressing PDGF induces hyperproliferation of progenitor cells and excessive, ectopic production of oligodendrocytes. However, the superfluous oligodendrocytes die at an immature stage of differentiation, leaving a normal complement of myelin-forming cells. Therefore, cell survival controls override proliferation controls for determining the final number and distribution of mature oligodendrocytes.

A novel organ culture model of a joint for the evaluation of static and dynamic load on articular cartilage.

OBJECTIVES: The purpose of this study was to create a novel ex vivo organ culture model for evaluating the effects of static and dynamic load on cartilage. METHODS: The metatarsophalangeal joints of 12 fresh cadaveric bovine feet were skinned and dissected aseptically, and cultured for up to four weeks. Dynamic movement was applied using a custom-made machine on six joints, with the others cultured under static conditions. Chondrocyte viability and matrix glycosaminoglycan (GAG) content were evaluated by the cell viability probes, 5-chloromethylfluorescein diacetate (CMFDA) and propidium iodide (PI), and dimethylmethylene blue (DMMB) assay, respectively. RESULTS: Chondrocyte viability in the static model decreased significantly from 89.9% (sd 2.5%) (Day 0) to 66.5% (sd 13.1%) (Day 28), 94.7% (sd 1.1%) to 80. 9% (sd 5.8%) and 80.1% (sd 3.0%) to 46.9% (sd 8.5%) in the superficial quarter, central half and deep quarter of cartilage, respectively (p < 0.001 in each zone; one-way analysis of variance). The GAG content decreased significantly from 6.01 µg/mg (sd 0.06) (Day 0) to 4.71 µg/mg (sd 0.06) (Day 28) (p < 0.001; one-way analysis of variance). However, with dynamic movement, chondrocyte viability and GAG content were maintained at the Day 0 level over the four-week period without a significant change (chondrocyte viability: 92.0% (sd 4.0%) (Day 0) to 89.9% (sd 0.2%) (Day 28), 93.1% (sd 1.5%) to 93.8% (sd 0.9%) and 85.6% (sd 0.8%) to 84.0% (sd 2.9%) in the three corresponding zones; GAG content: 6.18 µg/mg (sd 0.15) (Day 0) to 6.06 µg/mg (sd 0.09) (Day 28)). CONCLUSION: Dynamic joint movement maintained chondrocyte viability and cartilage GAG content. This long-term whole joint culture model could be of value in providing a more natural and controlled platform for investigating the influence of joint movement on articular cartilage, and for evaluating novel therapies for cartilage repair.Cite this article: Y-C. Lin, A. C. Hall, A. H. R. W. Simpson. A novel organ culture model of a joint for the evaluation of static and dynamic load on articular cartilage. Bone Joint Res 2018;7:205-212. DOI: 10.1302/2046-3758.73.BJR-2017-0320.

A potential key role for alpha-haemolysin of Staphylococcus aureus in mediating chondrocyte death in septic arthritis.

OBJECTIVES: Staphylococcus aureus (S. aureus) is the most commonly implicated organism in septic arthritis, a condition that may be highly destructive to articular cartilage. Previous studies investigating laboratory and clinical strains of S. aureus have demonstrated that potent toxins induced significant chondrocyte death, although the precise toxin or toxins that were involved was unknown. In this study, we used isogenic S. aureus mutants to assess the influence of alpha (Hla)-, beta (Hlb)-, and gamma (Hlg)-haemolysins, toxins considered important for the destruction of host tissue, on in situ bovine chondrocyte viability. METHODS: Bovine cartilage explants were cultured with isogenic S. aureus mutants and/or their culture supernatants. Chondrocyte viability was then assessed within defined regions of interest in the axial and coronal plane following live- and dead-cell imaging using the fluorescent probes 5-chloromethylfluorescein diacetate and propidium iodide, respectively, and confocal laser-scanning microscopy. RESULTS: Hla-producing mutants caused substantial chondrocyte death compared with the toxin-deficient control (Hla-Hlb-Hlg-), whilst mutants producing Hlb and Hlg in the absence of Hla induced minimal chondrocyte death. Coronal studies established that Hla-induced chondrocyte death started in the superficial zone of cartilage and spread to deeper layers, whereas Hlb and Hlg toxins were without significant effect. CONCLUSION: This study identified Hla as a highly potent S. aureus toxin that caused rapid chondrocyte death in bovine cartilage, with other toxins or metabolic products produced by the bacteria playing a minor role. The identification of Hla in mediating chondrocyte death may assist in the development of therapeutic strategies aimed at reducing the extent of cartilage damage during and after an episode of septic arthritis.Cite this article: I. D. M. Smith, K. M. Milto, C. J. Doherty, S. G. B. Amyes, A. H. R. W. Simpson, A. C. Hall. A potential key role for alpha-haemolysin of Staphylococcus aureus in mediating chondrocyte death in septic arthritis. Bone Joint Res 2018;7:457-467. DOI: 10.1302/2046-3758.77.BJR-2017-0165.R1.

Drying of open animal joints in vivo subsequently causes cartilage degeneration.

OBJECTIVES: During open orthopaedic surgery, joints may be exposed to air, potentially leading to cartilage drying and chondrocyte death, however, the long-term effects of joint drying in vivo are poorly understood. We used an animal model to investigate the subsequent effects of joint drying on cartilage and chondrocytes. METHODS: The patellar groove of anaesthetised rats was exposed (sham-operated), or exposed and then subjected to laminar airflow (0.25m/s; 60 minutes) before wounds were sutured and animals recovered. Animals were monitored for up to eight weeks and then sacrificed. Cartilage and chondrocyte properties were studied by histology and confocal microscopy, respectively. RESULTS: Joint drying caused extensive chondrocyte death within the superficial regions of cartilage. Histology of dried cartilage demonstrated a loss of surface integrity at four weeks, fibrillations at eight weeks, and an increased modified Mankin score (p < 0.001). Cartilage thickness increased (p < 0.001), whereas chondrocyte density decreased at four weeks (p < 0.001), but then increased towards sham-operated levels (p < 0.01) at eight weeks. By week eight, chondrocyte pairing/clustering and cell volume increased (p < 0.05; p < 0.001, respectively). CONCLUSIONS: These in vivo results demonstrated for the first time that as a result of laminar airflow, cartilage degeneration occurred which has characteristics similar to those seen in early osteoarthritis. Maintenance of adequate cartilage hydration during open orthopaedic surgery is therefore of paramount importance.Cite this article: Dr A. Hall. Drying of open animal joints in vivo subsequently causes cartilage degeneration. Bone Joint Res 2016;5:137-144. DOI: 10.1302/2046-3758.54.2000594.

The volume changes associated with the operation of the 'simple' transporter.

The effects of hydrostatic pressure (0.1-50 MPa) on uridine transport mediated by the 'simple' facilitated nucleoside transporter of guinea-pig and human erythrocytes have been studied in an attempt to identify the volume changes which occur during transport. Pressure inhibited the zero-trans (influx or efflux) mode of uridine transport in guinea-pig cells significantly more (about 2.2- x) than equilibrium exchange. The equilibrium binding of 3H-nitrobenzylthioinosine, a potent specific inhibitor of nucleoside transport, to human red cells and ghosts, was not significantly altered by pressure suggesting that the permeation site was unperturbed. Thus pressure inhibited the transporter primarily by preventing the volume increase associated with the translocation step. Furthermore, the return of the 'empty' transporter was found to be rate-limiting because it required a larger increase in volume than when the transporter was loaded with substrate.

Nature of the cation leak induced in erythrocyte membranes by Kanagawa haemolysin of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is an important enteric pathogen that produces an exotoxin prepared as Kanagawa haemolysin (KH). Isotope flux techniques were used to analyse toxin action on the basal permeability of human erythrocytes. KH induced a cation leak that was (i) rapid in onset (lag phase < 1 min), (ii) 'pore-like' in terms of kinetic characteristics, and (iii) of high magnitude initially (first 10 min) and then subsequently lower (but still raised with reference to control cells). The susceptibilities of the induced flux pathway to washout in initial and later periods suggested a protracted binding time course for toxin action. Neuraminidase treatment of erythrocytes enhanced both haemolysis and flux induced by KH, suggesting that the affinity of the toxin for the membrane had increased, possibly as a result of additional toxin receptors being unmasked by this enzyme. These results show that KH elevates the basal permeability of human erythrocytes in a complex manner, a process that probably underlies the deleterious effects of this toxin on cellular function.

Evidence for the presence of volume-sensitive KCl transport in 'young' human red cells.

'Young' human red cells are shown to possess a specific K+ pathway which is dependent on Cl- and sensitive to cell volume. This system was latent in 'mature' cells but was revealed by high hydrostatic pressure. This suggests the pathway is functionally active in 'young' cells but becomes masked with cell maturation.

Use of non-degenerate human osteochondral tissue and confocal laser scanning microscopy for the study of chondrocyte death at cartilage surgery.

Although autologous osteochondral grafting has been widely applied in humans, most in vitro work has been on animal models. The aims of this study were to: (i) elaborate a full thickness human femoral condylar model using discard material from knee arthroplasty resections, and (ii) use this model to assess chondrocyte viability in response to surgical trauma. Homogeneous regions of human lateral femoral condyle bone-cartilage were procured from knee arthroplasty resections. These were graded prospectively, firstly by visual inspection, and then by confocal laser scanning microscopy (CLSM). Samples were subjected to tests of tissue hydration, including analysis of water content and swelling after excision from underlying bone. Surgical cuts were made in explants that were macroscopically and microscopically normal (i.e. Grade 0). Associated margins of death were assessed from both transverse and surface perspectives. Thirty-nine samples were obtained from anterior and distal femoral cuts (16 knees from 13 patients) for (1) macroscopic grading, (2) microscopic analysis, (3) analyses of water content as cut and on re-equilibration after excision from bone. Thirteen were Grade 0 on macroscopic viewing--however one showed fibrillation on microscopy and was therefore reassigned Grade 1. Grade 0 tissue had a water content of 73.8+/-0.38%, in agreement with control values from the literature. Tissues of Grades 2 and 3 were significantly (P=0.03, and P=0.004) more hydrated (76.0+/-0.59%, 76.7+/-0.99%) than Grade 0 tissue. Grade 0 tissue from the anterior cut did not swell significantly following excision from subchondral bone. However Grade 0 tissue from the distal cut showed a small but statistically significant (P=0.019) increase in water content (1.68+/-0.39%) following excision. With increasing grade there was increased tendency to swell off the bone (P<0.0001). Transverse imaging showed that the Acufex MP surgical harvester caused a greater margin of cell death (211+/-18.3 microm) in the superficial zone (SZ) than the mid-zone (50.5+/-13.6 microm; P=0.022), or SZ death from a scalpel cut (33.0+/-8.5 microm; P=0.0009). Similarly, in unfixed samples viewed from the surface perspective, the margin of death for the surgical harvester (277+/-7.2 microm) was significantly (P<0.0001) greater than that for the scalpel (38.8+/-7.2 microm). If macroscopically and microscopically non-degenerate, then human lateral femoral condylar cartilage from the anterior cut of knee resections has normal biophysical parameters (water content and lack of swelling on excision). The surgical harvester (Acufex), used in human osteochondral grafting, caused a substantial margin of cell death at the periphery of the graft, and the SZ appeared to be especially vulnerable. This effect may be important in terms of limiting the reparative capacity of the SZ. The harmful effect on viability is likely to impede lateral repair which is fundamental to subsequent structural and functional integrity.

Chondrocyte death associated with human femoral osteochondral harvest as performed for mosaicplasty.

BACKGROUND: Autologous osteochondral transfer is an option for the treatment of articular defects. However, there are concerns about graft integration and the nature of the tissue forming the cartilage-cartilage bridge. Chondrocyte viability at graft and recipient edges is thought to be an important determinant of the quality of repair. The purpose of the present study was to evaluate early cell viability at the edges of osteochondral grafts from ex vivo human femoral condyles. METHODS: Fresh human tissue was obtained from eleven knees at the time of total knee arthroplasty for the treatment of osteoarthritis. Osteochondral cylinders were harvested with use of a 4.5-mm-diameter mosaicplasty osteotome from regions of the anterolateral aspect of the femoral condyle that were macroscopically nondegenerate and histologically nonfibrillated. Plugs were assessed for marginal cell viability by means of confocal laser scanning microscopy. RESULTS: The diameter of the cartilaginous portion of the osteochondral plugs was a mean (and standard error of the mean) of 4.84 +/- 0.12 mm (as determined on the basis of three plugs). This value was approximately 300 microm greater than the measured internal diameter of the osteotome. There was a substantial margin of superficial zone cell death (mean thickness, 382 +/- 68.2 microm), with >99% cell viability seen more centrally (as determined on the basis of five plugs). Demiplugs were created by splitting the mosaicplasty explants with a fresh number-11 scalpel blade. The margin of superficial zone cell death at the curved edge was significantly greater than that at the site of the scalpel cut (390.3 +/- 18.8 microm compared with 34.8 +/- 3.2 microm; p = 0.0286). Similar findings were observed when the cartilage alone was breached and the bone was left intact, with the margin of superficial zone cell death being significantly greater than that obtained in association with the straight scalpel incision (268 +/- 38.9 microm compared with 41.3 +/- 13.4 microm; p = 0.0286). The margin of superficial zone cell death showed no increase during the time-period between fifteen minutes and two hours after plug harvest. A mathematical approximation of the mosaicplasty region suggested that early cell death of this magnitude affects about one third of the superficial graft area. CONCLUSIONS: The results of the present study suggest that mosaicplasty, while capable of transposing viable hyaline cartilage, is associated with an extensive margin of cell death that is likely to compromise lateral integration and articular reconstruction.

Histamine phase shifts the hamster circadian pacemaker via an NMDA dependent mechanism.

The SCN acts as the central pacemaker for circadian rhythms in mammals. Histamine has been shown to affect circadian rhythms both in vivo and in vitro. We investigated the mechanism by which histamine phase shifts circadian rhythms in vitro. Hypothalamic slices containing the SCN were prepared from golden hamsters, and spontaneous firing rates of individual cells were recorded on the second day in vitro. Application of histamine (1 microM-10 mM) at the extrapolated time of 2 h after lights off (ZT 14) on day 1 in vitro delayed the time of peak firing in a dose-dependent manner. Pre-exposure to the N-methyl-D-aspartate (NMDA) receptor antagonist (+/-)-2-amino-5-phosphonopentanoic acid (AP-5; 100 microM-1 mM) 5 min before histamine (1 microM) was applied to the slice blocked the phase-delaying effects of histamine. Application of the H1 blocker mepryamine (100 nM) or the H2 blocker cimetidine (10 microM) followed by histamine had no effect on the phase delay induced by histamine. In whole cell recordings from acutely dissociated neurons of hamster SCN, histamine (50 microM) was shown to potentiate NMDA-evoked currents by 52 +/- 12%. These experiments demonstrate that histamine phase shifts of the circadian clock are dependent on NMDA receptor activation and that histamine can directly potentiate NMDA currents in SCN neurons. Histamine may alter circadian clock function by acting directly on NMDA receptors, possibly via binding to the polyamine site.

Suprachiasmatic nucleus neurons are glucose sensitive.

The suprachiasmatic nucleus (SCN) in the hypothalamus serves as the pacemaker for mammalian circadian rhythms. In a hamster brain slice preparation, the authors were able to record spontaneous activity from SCN cells for up to 4 days in vitro and verify a self-sustained rhythm in firing. The phase of this rhythm was altered by the concentration of glucose in the bathing medium, with time of peak firing advanced for a 20 mM glucose condition and slightly delayed for a 5 mM glucose condition, relative to 10 mM. The advancing effect of 20 mM glucose and the delaying effect of 5 mM glucose were not maintained during a 2nd day in vitro after changing the bathing medium back to 10 mM glucose, thus indicating the effect was not a permanent phase shift of the underlying oscillation. In experiments recording from cell-attached membrane patches on acutely dissociated hamster SCN neurons, exchanging the bathing medium from high (20 mM) to zero glucose increased potassium (K+)-selective channel activity. With inside-out membrane patches, the authors revealed the presence of a glybenclamide-sensitive K+ channel (190 pS) and a larger conductance (260 pS) Ca(2+)-dependent K+ channel that were both reversibly inhibited by ATP at the cytoplasmic surface. Furthermore, 1 mM tetraethylammonium chloride was demonstrated to advance peak firing time in the brain slice in a similar manner to a high concentration of glucose (20 mM). The authors interpret the result to imply that SCNs are sensitive to glucose, most probably via ATP modulation of K+ channel activity in these neurons. Tonic modulation of K+ channel activity appears to alter output of the pacemaker but does not reset the phase.

A multi-centre audit of epistaxis management in England: is there a case for a national review of practice?

OBJECTIVE: The ENT-UK Clinical Audit and Practice Advisory Group initiated a pilot audit to investigate variance in epistaxis management between six units nationwide. METHOD: All patients with a diagnosis of epistaxis who were admitted for in-patient care at six ENT departments between November 2011 and February 2012 were prospectively enrolled. RESULTS: A total of 166 patients were included in the study. Variance was demonstrated between the six units in a number of the key outcome areas. Twenty-eight per cent of patients were identified as eligible for operative intervention for epistaxis in one unit, compared with only 12.5 per cent in another. CONCLUSION: There are measurable, patient-relevant outcomes to assess epistaxis management and these can highlight areas of potential improvement. This pilot audit gives a snapshot of modern practice, which shows variance between the six units assessed. A national audit may allow us to improve patient experience and maximise efficiency in delivering emergency care in our most common patient encounter.