SLE mortality remains disproportionately high, despite improvements over the last decade.

Despite a marked improvement in 10-year survival for systemic lupus erythematosus (SLE) patients over the past five decades, mortality rates from SLE remain high compared to those in the general population. SLE was also among the leading causes of death in young women in the United States during 2000-2015. However, it is encouraging that SLE mortality rates and the ratios of SLE mortality rates to non-SLE mortality rates have decreased every year since the late 1990s. Despite this improvement, disparities in SLE mortality persist according to sex, race, age, and place of residence. Furthermore, demographic and geographic variables seem to modify the effect of each other in influencing SLE mortality, leading to interactions between sex/race/ethnicity-associated factors and geographic differences. In other words, individuals of the same sex/race/ethnicity had differences in SLE mortality depending on where they lived. These observations highlight SLE as an important public health issue. The recognition of SLE as a leading cause of death in the general population might spur targeted public health programs and research funding to address the high lupus mortality.

ESOPHAGEAL ATRESIA WITH RECURRENT TRACHEOESOPHAGEAL FISTULAS AND MICRODUPLICATION 22q11.23.

The microduplication 22q11.2 syndrome has a wide range of clinical manifestations. The phenotype ranges from normal to mental retardation and congenital anomalies. Esophageal atresia/tracheoesophageal fistula (EA/TEF) has recently been linked with the Tbx1 gene mutation located on the long arm of chromosome 22(22q11.21). We report a case with 1.4 Mb 22q11.23 duplication detected by array-CGH. The father of this infant has the same interstitial microduplication but with a normal phenotype. The phenotype seen in our case is type C (3B) esophageal atresia, tracheoesophageal fistula, and ventricular septal defect. Our patient underwent primary repair of OA/TEF malformations, which was later complicated by pneumonia and a recurrent TEF.

Elevated levels of leukotriene C4 synthase mRNA distinguish a subpopulation of eosinophilic oesophagitis patients.

BACKGROUND: Cysteinyl leukotrienes contribute to Th2-type inflammatory immune responses. Their levels in oesophageal tissue, however, do not distinguish patients with eosinophilic oesophagitis (EoE) from controls. OBJECTIVE: We asked whether mRNA levels of leukotriene C4 synthase (LTC4 S), a key regulator of leukotriene production, could serve as a marker for EoE. METHODS: Digital mRNA expression profiling (nCounter(®) Technology) was performed on proximal and distal oesophageal biopsies of 30 paediatric EoE patients and 40 non-EoE controls. Expression data were confirmed with RT-qPCR. LTC4 S mRNA levels were quantified in whole blood samples. Leukotriene E4 was measured in urine. RESULTS: LTC4 S mRNA levels were elevated in proximal (2.6-fold, P < 0.001) and distal (2.9-fold, P < 0.001) oesophageal biopsies from EoE patients. Importantly, increased LTC4 S mRNA transcripts identified a subpopulation of EoE patients (28%). This patient subgroup had higher serum IgE levels (669 U/mL vs. 106 U/mL, P = 0.01), higher mRNA transcript numbers of thymic stromal lymphopoietin (TSLP) (1.6-fold, P = 0.009) and CD4 (1.4-fold, P = 0.04) but lower IL-23 mRNA levels (0.5-fold, P = 0.04). In contrast, elevated levels of IL-23 mRNA were found in oesophageal biopsies of patients with reflux oesophagitis. LTC4 S mRNA transcripts in whole blood and urinary excretion of leukotriene E4 were similar in EoE patient subgroups and non-EoE patients. CONCLUSION & CLINICAL RELEVANCE: Elevated oesophageal expression of LTC4 S mRNA is found in a subgroup of EoE patients, concomitant with higher serum IgE levels and an oesophageal transcriptome indicative of a more-pronounced allergic phenotype. Together with TSLP and IL-23 mRNA levels, oesophageal LTC4 S mRNA may facilitate diagnosis of an EoE subpopulation for personalized therapy.

Acute food deprivation-induced relapse to heroin seeking after short and long punishment-imposed abstinence in male rats.

RATIONAL: Stress is a major trigger for drug relapse in humans and animal models, even after prolonged abstinence. However, animal models for stress-induced relapse were criticized for the lack of predictive and face validity. OBJECTIVES: Here we investigated the effect of acute food deprivation stress in a novel stress-induced relapse model using voluntary, punishment-imposed abstinence from heroin. We also performed a detailed characterization of the development of punishment-imposed abstinence. METHODS: Male rats were trained to self-administered heroin (0.1 mg/kg/infusion) for 2 weeks, using the seeking-taking chained schedule. Pressing the 'seeking' lever led to the insertion of the 'taking' lever and pressing the take lever resulted in heroin infusion. Following self-administration training, rats were exposed to 8 or 21 days of heroin-seeking punishment. During punishment, 30% of the completed seek links resulted in a mild escalating footshock instead of take lever presentation. Next, rats were tested for heroin seeking under extinction conditions after 24 h of food deprivation and sated conditions. RESULTS: Probabilistic punishment of seeking lever responses resulted in gradual suppression of heroin seeking and taking. Exposure to food-deprivation stress induced a robust relapse to heroin seeking after short and long punishment-imposed abstinence periods, without significant effects of time, i.e., no incubation of heroin seeking. Individual differences were observed in the development of punishment-induced abstinence and stress-induced relapse. CONCLUSIONS: These results suggest that stress is a reliable trigger to relapse even after a prolonged period of punishment-induced, voluntary abstinence.

Performance of the new biological small- and wide-angle X-ray scattering beamline 13A at the Taiwan Photon Source.

Recent developments in the instrumentation and data analysis of synchrotron small-angle X-ray scattering (SAXS) on biomolecules in solution have made biological SAXS (BioSAXS) a mature and popular tool in structural biology. This article reports on an advanced endstation developed at beamline 13A of the 3.0 GeV Taiwan Photon Source for biological small- and wide-angle X-ray scattering (SAXS-WAXS or SWAXS). The endstation features an in-vacuum SWAXS detection system comprising two mobile area detectors (Eiger X 9M/1M) and an online size-exclusion chromatography system incorporating several optical probes including a UV-Vis absorption spectrometer and refractometer. The instrumentation and automation allow simultaneous SAXS-WAXS data collection and data reduction for high-throughput biomolecular conformation and composition determinations. The performance of the endstation is illustrated with the SWAXS data collected for several model proteins in solution, covering a scattering vector magnitude q across three orders of magnitude. The crystal-model fittings to the data in the q range ∼0.005-2.0 Å-1 indicate high similarity of the solution structures of the proteins to their crystalline forms, except for some subtle hydration-dependent local details. These results open up new horizons of SWAXS in studying correlated local and global structures of biomolecules in solution.

Host cell protein profiling of commercial therapeutic protein drugs as a benchmark for monoclonal antibody-based therapeutic protein development.

Therapeutic proteins including monoclonal antibodies (mAbs) are usually produced in engineered host cell lines that also produce thousands of endogenous proteins at varying levels. A critical aspect of the development of biotherapeutics manufacturing processes is the removal of these host cell proteins (HCP) to appropriate levels in order to minimize risk to patient safety and drug efficacy. During the development process and associated analytical characterization, mass spectrometry (MS) has become an increasingly popular tool for HCP analysis due to its ability to provide both relative abundance and identity of individual HCP and because the method does not rely on polyclonal antibodies, which are used in enzyme-linked immunosorbent assays. In this study, HCP from 29 commercially marketed mAb and mAb-based therapeutics were profiled using liquid chromatography (LC)-MS/MS with the identification and relative quantification of 79 individual HCP in total. Excluding an outlier drug, the relative levels of individual HCP determined in the approved therapeutics were generally low, with an average of 20 ppm (µmol HCP/mol drug) measured by LC-MS/MS, and only a few (<7 in average) HCP were identified in each drug analyzed. From this analysis, we also gained knowledge about which HCP are frequently identified in mAb-based products and their typical levels relative to the drugs for the identified individual HCP. In addition, we examined HCP composition from antibodies produced in house and found our current development process brings HCP to levels that are consistent with marketed drugs. Finally, we described a specific case to demonstrate how the HCP information from commercially marketed drugs could inform future HCP analyses.

Augmented Reality in Pediatric Septic Shock Simulation: Randomized Controlled Feasibility Trial.

BACKGROUND: Septic shock is a low-frequency but high-stakes condition in children requiring prompt resuscitation, which makes it an important target for simulation-based education. OBJECTIVE: In this study, we aimed to design and implement an augmented reality app (PediSepsisAR) for septic shock simulation, test the feasibility of measuring the timing and volume of fluid administration during septic shock simulation with and without PediSepsisAR, and describe PediSepsisAR as an educational tool. We hypothesized that we could feasibly measure our desired data during the simulation in 90% of the participants in each group. With regard to using PediSepsisAR as an educational tool, we hypothesized that the PediSepsisAR group would report that it enhanced their awareness of simulated patient blood flow and would more rapidly verbalize recognition of abnormal patient status and desired management steps. METHODS: We performed a randomized controlled feasibility trial with a convenience sample of pediatric care providers at a large tertiary care pediatric center. Participants completed a prestudy questionnaire and were randomized to either the PediSepsisAR or control (traditional simulation) arms. We measured the participants' time to administer 20, 40, and 60 cc/kg of intravenous fluids during a septic shock simulation using each modality. In addition, facilitators timed how long participants took to verbalize they had recognized tachycardia, hypotension, or septic shock and desired to initiate the sepsis pathway and administer antibiotics. Participants in the PediSepsisAR arm completed a poststudy questionnaire. We analyzed data using descriptive statistics and a Wilcoxon rank-sum test to compare the median time with event variables between groups. RESULTS: We enrolled 50 participants (n=25 in each arm). The timing and volume of fluid administration were captured in all the participants in each group. There was no statistically significant difference regarding time to administration of intravenous fluids between the two groups. Similarly, there was no statistically significant difference between the groups regarding time to verbalized recognition of patient status or desired management steps. Most participants in the PediSepsisAR group reported that PediSepsisAR enhanced their awareness of the patient's perfusion. CONCLUSIONS: We developed an augmented reality app for use in pediatric septic shock simulations and demonstrated the feasibility of measuring the volume and timing of fluid administration during simulation using this modality. In addition, our findings suggest that PediSepsisAR may enhance participants' awareness of abnormal perfusion.

First evidence of a possible association between gastric acid suppression during pregnancy and childhood asthma: a population-based register study.

BACKGROUND: Recent data in mice suggest that acid suppression during pregnancy yields offspring with type 2 T helper-dominant immunity, suggesting a predisposition for allergy. OBJECTIVE: To determine the association of in utero exposure to acid-suppressive medications and the subsequent development of allergic diseases in children. METHODS: We studied a population-based observational cohort formed by linking data from three Swedish national healthcare registers: the Medical Birth Register, the Hospital Discharge Register, and the Swedish Prescribed Drug Register. Main outcome measures included a hospital discharge diagnosis of an allergic disease or prescription for asthma medications, epinephrine auto-injectors, antihistamines or steroid ointments in children. Data were analysed using the Mantel-Haenszel procedure. RESULTS: Twenty-nine thousand four hundred and ninety (5.03%) children had a discharge diagnosis of allergy or prescriptions for allergy medications. Five thousand six hundred and forty-five (0.96%) children had been exposed to acid suppression therapy during pregnancy; of these, 405 (0.07%) were treated for allergic diseases. Exposure to acid-suppressive medications in utero was associated with an increased odds ratio (OR) for developing allergy (OR 1.43, 95% confidence interval (95% CI) 1.29-1.59). We observed this association irrespective of the type of drug, time of exposure during pregnancy, and maternal history of allergy. The use of maternal acid-suppressive medication was associated with an increased OR for the development of childhood asthma (3.7% in the population at large vs. 5.6% in exposed children, OR 1.51, 95% CI 1.35-1.69), but not for other allergic diseases. CONCLUSION: These data provide first evidence of a significant association between in utero exposure to acid-suppressive drugs and the risk of developing childhood asthma.

Exhaled breath condensate nitrite in premature infants with bronchopulmonary dysplasia.

BACKGROUND: Tracheal aspirate is the conventional method to measure biomarkers of inflammation and oxidation from premature infants on mechanical ventilation at risk for bronchopulmonary dysplasia (BPD), but this method is invasive. Exhaled breath condensate (EBC) is a novel, non-invasive method that has been used in older populations. Nitrite, a stable metabolite of nitric oxide (NO), is elevated in inflammatory conditions. We aim to investigate the feasibility of EBC nitrite collection from ventilated premature infants and to quantify EBC nitrite in infants with and without BPD. We hypothesize that EBC nitrite correlates with TA nitrite, and that EBC nitrite in the first week of life is higher in infants who will develop BPD than those without BPD. METHODS: In a pilot prospective cohort study, TA and EBC were collected in the first week of life from mechanically ventilated premature infants. Nitrite levels were measured using chemiluminescence. RESULTS: EBC nitrite significantly correlated with TA nitrite (r = 0.45, p = 0.025). Of 40 infants, 33 (82.5%) developed BPD. EBC and TA nitrite levels collected in the first week of life had a higher trend in infants with BPD than those without BPD (p = 0.23 and 0.38 respectively). CONCLUSIONS: Higher trend of EBC nitrite in the first week of life was associated with the development of BPD. Correlation of nitrite level in EBC with that in TA (conventional method) highlights the utility of EBC as an alternative, non-invasive method to measure inflammation. Further refinement of conditions and timing may optimize the predictive value of EBC nitrite.

Randomised clinical trial: a phase 1, dose-ranging study of the anti-matrix metalloproteinase-9 monoclonal antibody GS-5745 versus placebo for ulcerative colitis.

BACKGROUND: Matrix metalloproteinase-9 is a proteolytic enzyme whose expression is increased in ulcerative colitis. AIM: To evaluate the safety and efficacy of GS-5745, a fully humanised anti-matrix metalloproteinase-9 monoclonal antibody, in moderately-to-severely active ulcerative colitis. METHODS: We randomised 74 patients with ulcerative colitis to treatment with single or multiple ascending intravenous or subcutaneous doses of GS-5745 or placebo. Multiple-dose cohorts received either IV infusions (0.3, 1.0, 2.5 or 5.0 mg/kg GS-5745 or placebo) every 2 weeks (three total IV infusions) or five weekly SC injections (150 mg GS-5745 or placebo). The primary outcomes were the safety, tolerability and pharmacokinetics of escalating single and multiple doses of GS-5745. Exploratory analyses in the multiple-dose cohorts included clinical response (≥3 points or 30% decrease from baseline in Mayo Clinic score and ≥1 point decrease in the rectal bleeding subscore or a rectal bleeding subscore ≤1) and clinical remission (a complete Mayo Clinic score ≤2 with no subscore >1) at Day 36. Biological effects associated with a clinical response to GS-5745 were explored using histological and molecular approaches. RESULTS: Twenty-three of the 42 patients (55%) receiving multiple doses of GS-5745 had adverse events, compared with 5/8 patients (63%) receiving placebo. GS-5745 showed target-mediated drug disposition, approximately dose-proportional increases in maximum plasma concentration and more than dose-proportional increases in the area under the plasma drug concentration-time curve. Clinical response occurred in 18/42 patients (43%) receiving GS-5745 compared with 1/8 patients (13%) receiving placebo. Clinical remission occurred in 6/42 patients (14%) receiving GS-5745 and 0/8 (0%) receiving placebo. Patients with a clinical response to GS-5745 had reductions in matrix metalloproteinase-9 tissue levels (mean 48.9% decrease from baseline compared with a mean 18.5% increase in nonresponders, P = 0.008) significant improvements in histopathology scores (confirmed with three separate histological disease activity indices), as well as changes in colonic gene expression that were consistent with reduced inflammation. CONCLUSION: This phase 1 trial provides preliminary evidence for the safety and therapeutic potential of GS-5745 in the treatment of ulcerative colitis.

Stimulation of signal transduction pathways in osteoblasts by mechanical strain potentiated by parathyroid hormone.

Second-messenger systems have been implicated to transmit mechanical stimulation into cellular signals; however, there is no information on how mechanical stimulation is affected by such systemic factors as parathyroid hormone (PTH). Regulation of adenylyl cyclase and phosphatidylinositol pathways in rat dentoalveolar bone cells by mechanical strain and PTH was investigated. Two different cell populations were isolated after sequential enzyme digestions from dentoalveolar bone (group I and group II) to study potential differences in response. Mechanical strain was applied with 20 kPa of vacuum intermittently at 0.05 Hz for periods of 0.5, 1, 5, 10, and 30 minutes and 1, 3, and 7 days using the Flexercell system. Levels of cAMP, measured by RIA, and levels of inositol 1,4,5-triphosphate (IP3) and protein kinase C activity (PKC), measured by assay systems, increased with mechanical strain. When PTH was added to the cells, there was a significant increase in levels of all the intracellular signals, which appeared to potentiate the response to mechanical strain. IP3 levels (0.5 minute) peaked before those of PKC activity (5 minutes), which in turn peaked before those of cAMP (10 minutes). Group II cells showed higher levels of cAMP and IP3 than the group I cells. This suggests that the former may ultimately play the predominant roles in skeletal remodeling in response to strain. Immunolocalization of the cytoskeleton proteins vimentin and alpha-actinin, focal contact protein vinculin, and PKC showed a marked difference between strained and nonstrained cells. However, the addition of PTH did not cause any significant effect in cytoskeleton reorganization. Staining of PKC and vimentin, alpha-actinin, and vinculin suggests that PKC participates actively in the transduction of mechanical signals to the cell through focal adhesions and the cytoskeleton, although only PKC seemed to change with short time periods of strain. In conclusion, dentoalveolar osteoblasts responded to mechanical strain initially through increases in levels of IP3, PKC activity, and later cAMP, and this response was potentiated when PTH was applied together with mechanical strain.

The effect of sutural growth rate on collagen phenotype synthesis.

We determined the ratio of newly-synthesized type III collagen to the total of type I and type III collagen in mouse interparietal sutural tissue at selected ages between birth and adulthood (36 weeks old). We incubated mouse calvaria explants in Trowell-type organ culture dishes for one h and then added [14C]-glycine for two h. We dissected the interparietal sutural tissues for collagen solubilization by limited pepsin digestion. Fluorographic visualization of separated radiolabeled collagens, after SDS-PAGE, found the ratio of collagen type III alpha-chains to the total type I and type III alpha-chains to be age-dependent. The proportion of type III alpha-chains at birth was quite high, but there was a significant drop (p less than 0.05) during the first two days of life, probably because of the sudden environmental change from in utero. The proportion of type III alpha-chains rose significantly from day 2 to day 4, reaching a maximum and then dropping significantly to about the same proportion as at birth by day 7. A further significant drop took place during the second week of life, with the proportion stabilized at around 3.5% at two weeks to ten weeks of age. A final significant drop during the eleventh week of life led to no detectable synthesis of type III collagen after 12 weeks of age. The changes in the collagen phenotype ratio did not relate to changes in body weight during growth and development, which suggests that the interparietal suture may have an independent maturing pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of force level on synthesis of type III and type I collagen in mouse interparietal suture.

Nine-week-old Swiss male white mice were divided into groups killed after time intervals of force application of six h, and one, three, five, seven, ten, 14, 21, and 28 days. Each group had 45 animals: three control, three sham-operated, and three experimental animals for each of the five force levels: 50 g, 35 g, 25 g, 15 g, and 5 g. The experimental animals had helical springs placed surgically in their calvaria for expansion of the interparietal suture. The sham-operated animals received inactive springs. Control animals were at the same age as the experimental and sham-operated animals. After death, the amount of sutural expansion was measured, and the calvaria with the implanted springs were explanted into Trowell-type organ culture dishes. [14C]-glycine was added for two h after 60 min of culture for all explants. The rate of suture expansion was directly proportional to the force value of the tensile stress, and a maximum 2.0-mm expansion was achieved for all force levels by the 28th day. Sutural collagen was solubilized by limited pepsin digestion, and radiolabeled types I and III alpha-chains were separated by SDS-PAGE, visualized fluorographically, and measured densitometrically. All the experimental and sham-operated animals responded with a rapid rise followed by an almost equally rapid fall in the proportion of newly-synthesized type III collagen before becoming stabilized for the rest of the experimental period at a level that was significantly higher than that of the control and sham-treated animals of the same age.(ABSTRACT TRUNCATED AT 250 WORDS)

Collagen and prostaglandin synthesis in force-stressed periodontal ligament in vitro.

A periodontal organ culture system capable of receiving orthodontic type forces was developed. Histological, radioautographical, collagen, and prostaglandin synthetic data demonstrated the vitality of the organ over a 24-hour period of culture. Significant increases in the proportion of type III collagen synthesized during periods of active stress were found, but no alterations in relative levels of prostaglandins synthesized during periods of force application were discernible.

Continuous stressing of mouse interparietal suture fibroblasts in vitro.

The morphological and biochemical response of sutural fibroblasts in vitro to continuous force was examined. Cells from mouse interparietal sutures were grown and subcultured on glass slides. Titanium disks coated with collagen were allowed to attach to the cellular multilayers. Four of the glass slides were then placed at an angle of 75 degrees for a period of three days so that continuous stress would be created, while four others were left flat. Also, four glass slides were left flat with no disk. Following the incubation period, the dishes were labeled with 14C-glycine for 15 h. The cells and medium were then collected for collagen extraction followed by SDS-polyacrylamide gel electrophoresis. Dried gels impregnated with fluor were exposed to x-ray films that were then scanned densitometrically for collagen types I and III. It was found that the proportion of newly-synthesized type III collagen increased significantly with the application of continuous stress. A second set of experimental and control glass slides was fixed in glutaraldehyde and post-fixed in osmium tetroxide. Following critical-point drying and coating, the glass slides were examined under a scanning electron microscope. The scanning images showed the formation of a ligament-like structure between the disk and the glass slide. Moreover, mitotic activity, as evidenced by spheroidal cells, was stimulated in the areas previously adjacent to the disc, which had since moved away. This system offers a standardized continuous force system that can stress cells in a ligament-like structure and thus provides an in vitro model analogous to clinical orthodontic and orthopedic stress.

Complications and recommended practices for electrosurgery in laparoscopy.

BACKGROUND: Electrosurgery is one of the most commonly used energy systems in laparoscopic surgery. Two major categories of potential complications related to electrosurgery in laparoscopy are mechanical trauma and electrothermal injury. The latter can result from unrecognized energy transfer in the operational field or, less commonly, to unnoticed stray current outside the laparoscopic field of view. Stray current can result from insulation failure, direct coupling, or capacitive coupling. METHODS: We reviewed the literature concerning essential biophysics of electrosurgery, including electrosurgical waveform differentiation, tissue effect, and variables that determine tissue effect. The incidence of electrosurgical injuries and possible mechanisms responsible for the injuries are discussed. Different types of injuries may result in different clinical manifestations and histopathological findings. Gross and microscopic pathological check-ups of the injury sites may distinguish between different mechanisms, and thus provide further clues postoperatively. RESULTS: Several recommended practices are proposed to avoid electrosurgical injury laparoscopically. To achieve electrosurgical safety and to prevent electrosurgical injuries, the surgical team should have a good understanding of the biophysics of electrosurgery, the basis of equipment and general tissue effects, as well as the surgeon's spatial orientation and hand-eye coordination. Some intraoperative adjuvant procedures and newly developed safety devices have become available may aid to improve electrosurgical safety. CONCLUSIONS: Knowledge of the biophysics of electrosurgery and the mechanisms of electrosurgical injury is important in recognizing potential complications of electrosurgery in laparoscopy. Procedures for prevention, intraoperative adjuvant maneuvers, early recognition of the injury with in-time salvage treatment, and alertness to postoperative warning signs can help reduce such complications.

A radioautographic study of the effect of age on the protein-synthetic and bone-deposition activity in interparietal sutures of male white mice.

Groups of male white mice were killed at 4, 5, 6, 7, 8, 9 and 10 weeks of age 2 h after intraperitoneal injection with [3H]-proline. Radioautographic analysis of sections of the interparietal suture demonstrated significantly greater protein-synthetic activity in the para-osseous zones relative to the middle zone (p less than 0.01) and a plateau of lower protein-synthetic activity by 7-8 weeks of age (p less than 0.05). Groups of mice were selected at 4, 6, 8 and 10 weeks of age. Each mouse was injected intraperitoneally with [3H]-proline three times at one-week intervals. Sutural growth rate was determined from incremental lines revealed by radioautographs prepared from serial paraffin sections of the interparietal suture and demonstrated a stabilization of growth by 8 weeks of age. This, together with the grain counting data, suggested that a mouse of 7-8 weeks would provide a suitable model for experimental studies in sutural remodelling response without masking effects by normal growth.

The effect of growth on collagen and glycosaminoglycans in the articular disc of the rat temporomandibular joint.

Newly synthesized collagen and glycosaminoglycans (GAG) were studied in the temporomandibular discs of male Sprague-Dawley rats of 3-13 weeks of age. Each age group had eight animals and [14C]glycine or [3H]glucosamine were used to determine the proportion of newly synthesized type III to type I collagens or the proportion of different types of newly synthesized GAGs during 4 h of labelling in organ culture. Separation of newly synthesized collagen bands from rat disc by sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed a peak in type III at the ages of 7 and 8 weeks. Type III collagen synthesis and the rate of mandibular growth were strongly related through all ages studied. GAG chains were separated by cellulose-acetate electrophoresis. Calculation of disintegrations/min per mg of wet disc tissue for each GAG peak showed that hyaluronic acid (HA), chondroitin-6-sulphate and keratan sulphate/chondroitin-4-sulphate (KS/C4S) were the predominant molecules synthesized in the disc. There was also a steady increase in newly synthesized HA and C6S synthesis up to 6 and 7 weeks respectively. Proportions of newly synthesized C4S/KS, HA and C6S were significantly higher than those of other GAGs with respect to ageing. From these observations it appears that the articular disc shows more of the characteristics of cartilage, as evidenced by the increased amounts of C6S and KS/C4S during the mandibular growth spurt at the ages of 6 and 7 weeks, similar to that of type III collagen. There were also increased amounts of HA, suggesting that during 5-7 weeks of age the rat disc is undergoing more active remodelling. This study provides baseline data for further analysis of the effects of mechanical loading and trauma on articular disc responses.

Effect of strain on bone nodule formation by rat osteogenic cells in vitro.

The purpose of this study was to assess in vitro bone nodule formation by cells exposed to a range of microstrain, at a sub-optimal oscillation frequency for bone formation. Fetal rat calvarial cells experienced a Flexercell regimen within either FLEX I (deformable) or FLEX II (non-deformable) substrates. Cells in FLEX I plates were exposed to growth medium only; those in FLEX II plates were exposed to either growth medium only, or growth medium + 10(-7) M IGF-1. Cell numbers were assessed from 1 to 6 days. Other cells were exposed to the Flexercell regimen (-2 kPa, 0.05 Hz) for 1-3 (Group 1), 3-6 (Group 2), 1-9 (Group 3) or 10-15 (Group 4) days and were maintained, at other times, under standard conditions. After 21 days, nodules were counted within each well and within the compression, <999, 1000-4900, 5000-9999, 10,000-14,999 and 15,000-25,000 microstrain regions of the FLEX I membrane. Cyclic deformation inhibited cell numbers from 1 to 6 days, compared to control or IGF-1 groups (P<0.001). The number of nodules in Groups 2 and 4 were greater than Groups 1 or 3 (P<0.001), but not different from control or IGF-1 groups. Compression or tensile microstrain significantly affected nodule formation in all groups, with Group 4 producing more nodules than other groups in most microstrain regions. Thus, the number of bone nodules produced by osteogenic cell cultures exposed to cyclic deformation was significantly affected by the timing of initiation and the characteristics and magnitude of the deformation regimen.