The effect of experimental diabetes and glycaemic control on guided bone regeneration: histology and gene expression analyses.

OBJECTIVES: To investigate the effect of experimental diabetes and metabolic control on intramembranous bone healing following guided bone regeneration (GBR). MATERIAL AND METHODS: Ninety-three Wistar rats were allocated to three experimental groups, healthy (H), uncontrolled diabetes (D) and controlled diabetes (CD). Twenty one days following diabetes induction, a standardised 5-mm defect was created at the mid-portion of each parietal bone. In 75 animals (25H, 25D, 25CD), one defect was treated with an intracranial and extracranial membrane according to the GBR principle, and one defect was left empty (control); five animals per group were then randomly sacrificed at 3, 7, 15, 30 and 60 days and processed for decalcified histology. In 18 animals (6H, 6D, 6CD), both defects were treated according to the GBR principle; three animals from each group were then randomly sacrificed at 7 and 15 days of healing and employed for gene expression analysis. RESULTS: Application of the GBR therapeutic principle led to significant bone regeneration even in the D group. However, at 15 and 30 days, the osteogenesis process was impaired by uncontrolled diabetes, as shown by the significant reduction in terms of defect closure (38-42%) and newly formed bone (54-61%) compared to the healthy group. The comparison of the D vs. H group at 15 days of healing yielded the largest number of genes with significantly differential expression, among which various genes associated with the ossification process (bmp4, ltbp4, thra and cd276) were identified. CONCLUSIONS: Uncontrolled diabetes seems to affect early phases of the bone regeneration following GBR. A misregulation of genes and pathways related to cell division, energy production, inflammation and osteogenesis may account for the impaired regeneration process in D rats. Further studies are warranted to optimise the GBR process in this medically compromised patient population.

WONKA: objective novel complex analysis for ensembles of protein-ligand structures.

WONKA is a tool for the systematic analysis of an ensemble of protein-ligand structures. It makes the identification of conserved and unusual features within such an ensemble straightforward. WONKA uses an intuitive workflow to process structural co-ordinates. Ligand and protein features are summarised and then presented within an interactive web application. WONKA's power in consolidating and summarising large amounts of data is described through the analysis of three bromodomain datasets. Furthermore, and in contrast to many current methods, WONKA relates analysis to individual ligands, from which we find unusual and erroneous binding modes. Finally the use of WONKA as an annotation tool to share observations about structures is demonstrated. WONKA is freely available to download and install locally or can be used online at http://wonka.sgc.ox.ac.uk.

Transcriptional profiling of "guided bone regeneration" in a critical-size calvarial defect.

OBJECTIVES: Guided bone regeneration (GBR) is a commonly utilized surgical technique in the craniofacial region. The transcriptional mechanisms associated with this type of bone regeneration are not well understood. The aim of this study was to characterize the transcriptome associated with GBR of a critical-size calvarial defect in the rat. MATERIAL AND METHODS: Critical-size calvarial defects were created in six Wistar strain rats and treated according to the principles of GBR. The tissue filling the regenerating defect was harvested at 7 and 14 days. Total RNA was extracted and microarray analysis was carried out to identify the differences in the transcriptome between days 7 and 14. RESULTS: Gene ontology (GO) analysis of the genes up-regulated at day 7 showed that immature wound healing-related mechanisms, such as protein metabolism and cell proliferation, were up-regulated at this time point. Furthermore, the immuno-inflammatory process was also up-regulated at the earlier time point. In contrast, by day 14, GO groups consistent with wound maturation, such as extracellular matrix formation, anatomical structure development and cell differentiation, were up-regulated. Furthermore, the functionally important GO categories of skeletal development, ossification and bone mineralization were up-regulated at day 14. Genes of interest that belonged to this group and were up-regulated at day 14 included growth and differentiation factors (Bmp2, Bmp3, Tgfb3), extracellular matrix proteins (osteocalcin, osteomodulin, stenniocalcin 1) and transcription factors (Runx2, Sox6, Satb2). Furthermore, a number of genes associated with Tgfß/Bmp and Wnt signalling were also up-regulated. Besides skeletogenesis, genes associated with angiogenesis and neurogenesis were also up-regulated at day 14. CONCLUSIONS: The transcriptome associated with a maturing GBR-treated craniofacial bone defect is characterized by the down-regulation of the immuno-inflammatory response and up-regulation of skeletogeneis-, angiogenesis- and neurogenesis-associated genes. The Tgfß/Bmp and Wnt signalling pathways play an important role in the regenerative process.

In vivo gene expression profile of guided bone regeneration associated with a microrough titanium surface.

OBJECTIVES: To determine the gene expression profile characteristic of "guided bone regeneration" associated with a microrough titanium surface. MATERIAL AND METHODS: Critical-size calvarial defects were treated with the principle of "guided bone regeneration," whereby the extracranial barriers were either polished (SMO) or microrough (SLA) titanium disks. After 7 and 14 days, the contents of the regenerating defect were collected, RNA was extracted and microarray analysis was carried out. At each time point, the healing associated with the microrough surface was compared with that associated with the polished titanium surface. RESULTS: On comparing the SLA and SMO profiles, there were few genes different at day 7 (∼250), whereas there were a large number of genes different at day 14 (∼6500). At day 14, the list of genes that were differentially regulated in response to the SMO and SLA surfaces had an over-representation of genes associated with the functionally relevant gene ontology categories of regeneration, skeletogenesis, mesenchymal cell differentiation, angiogenesis and neurogenesis. There were a greater number of genes within each of these functionally relevant categories that were up-regulated on the SLA surface compared with the SMO surface. The main signalling pathway that was differentially regulated between the two surfaces at day 14 was the Wnt signaling pathway. CONCLUSIONS: Minimal difference was observed between the SMO and the SLA samples at day 7, whereas significant differences were noted at day 14, including genes associated with a number of functionally relevant gene ontology groups. The differentially regulated biological processes provide an insight into the influence of surface topography on "guided bone regeneration" at the cellular and molecular level.

Narcolepsy in the custodial setting.

A case of a detained person who suffered from narcolepsy is presented. The management of the condition in general and with particular reference to the custodial setting is discussed. The management of obstructive sleep apnoea, the commonest sleep disorder, is contrasted. Forensic physicians are often confronted with rare or unusual medical problems in the custodial setting, often having to make immediate medical decisions without access to specialist medical advice. Twenty-four hour access to Internet medical resources in the custodial setting may be helpful.

Patterns of illicit drug use of prisoners in police custody in London, UK.

AIMS AND METHODS: The aims of the study were to explore the current characteristics of drug misusers seen in police custody and identify trends or changes that have taken place in the last decade. A prospective, anonymised, structured questionnaire survey was undertaken of consenting consecutive, self-admitted illicit drug users seen by forensic physicians in police custody within the Metropolitan Police Service in London, UK in 2003. RESULTS: 30% of detainees were dependent on heroin or crack cocaine. Drug users (n=113) were studied in 2003. 95.4% completed the questionnaire. 82% were male, 18% female. Mean age was 28.5 y (range 18-49). 80% were unemployed; significant mental health issues (e.g., schizophrenia) were present in 18%; 15% had alcohol dependence; heroin was the most frequently used drug (93%); crack cocaine -- 87%; mean daily cost of drugs -- heroin GBP 76 (range 20-240), crack GBP 81 (range 20-300). >50% users inject crack and heroin simultaneously. 56% used the intravenous route; 25% had shared needles; 100% had accessible sources of clean needles; 6.4% were hepatitis B positive; 42% were aware of hepatitis prophylaxis; hepatitis C positive -- 20.2%; 3.6% were HIV positive. Mean length of time of drug use was 7.5 y (range 1 month -- 20 years); 82% had served a previous prison sentence; 54% had used drugs in prison; 11% had used needles in prison; 3% of users stated they had started using in prison. 38% had been on rehabilitation programs; 11% had been on Drug Treatment and Testing; Orders (DTTO); 32% had used the services of Drug Arrest Referral Teams in police stations; 10% were in contact with Drug Teams at the time of assessment. CONCLUSIONS: In the last decade, there appears to be a substantial increase in the prevalence of drug use in this population -- particularly of crack cocaine. Treatment interventions are either not readily available, or not followed through. In very general terms, the illicit drug use problem appears to have significantly worsened in the population seen in police custody in London, UK, in the last decade although there is evidence that health education and harm reduction messages appear to have had some positive effects.

Binding constants of neuraminidase inhibitors: An investigation of the linear interaction energy method.

The linear interaction energy (LIE) method has been applied to the calculation of the binding free energies of 15 inhibitors of the enzyme neuraminidase. This is a particularly challenging system for this methodology since the protein conformation and the number of tightly bound water molecules in the active site are known to change for different inhibitors. It is not clear that the basic LIE method will calculate the contributions to the binding free energies arising from these effects correctly. Application of the basic LIE equation yielded an rms error with respect to experiment of 1.51 kcal mol(-1) for the free energies of binding. To determine whether it is appropriate to include extra terms in the LIE equation, a detailed statistical analysis was undertaken. Multiple linear regression (MLR) is often used to determine the significance of terms in a fitting equation; this method is inappropriate for the current system owing to the highly correlated nature of the descriptor variables. Use of MLR in other applications of the LIE equation is therefore not recommended without a correlation analysis being performed first. Here factor analysis was used to determine the number of useful dimensions contained within the data and, hence, the maximum number of variables to be considered when specifying a model or equation. Biased fitting methods using orthogonalized components were then used to generate the most predictive model. The final model gave a q(2) of 0.74 and contained van der Waals and electrostatic energy terms. This result was obtained without recourse to prior knowledge and was based solely on the information content of the data.

Biochemical risk factors in patients with renal staghorn stone disease.

Thirty-three patients operated on for renal staghorn calculous disease were studied retrospectively with respect to urine and stone composition, bacteriuria, and abnormalities of the urinary tract. Calcium phosphate was the most common stone constituent, present in 30 of 31 concrements. Twenty-one of these stones also contained magnesium ammonium phosphate, despite the fact that only 10 patients had presented evidence of urinary tract infection during the initial period of the disease. Twenty-four-hour urine composition was normal in only 3 patients. In 59 per cent an increased CaOx risk index was observed suggesting that CaOx risk factors might contribute to the development of staghorn concrements. A metabolic evaluation of staghorn stone formers appears to be of importance for design of the postoperative treatment.

Prevalence of HIV risk factors for individuals examined in clinical forensic medicine.

OBJECTIVE: to identify the prevalence of high-risk factors for infection with Human Immunodeficiency Virus (HIV) in individuals examined in clinical forensic medical practice and to determine opinions and attitudes about HIV in this patient group. DESIGN: Anonymised questionnaire completed by consecutive individuals seen in clinical forensic medical practice. SETTING: Police stations in London attended by Group IV forensic medical examiners. SUBJECTS: 518 individuals examined in police stations (including prisoners and suspects, those detained in police custody, police officers and victims of crime). RESULTS: 164 (31.7%) individuals did not respond to the questionnaire because of: 1) refusal (12.6%) 2) inability because of drugs and/or alcohol (11.4%) 3) mental illness/disorder (4.2%) or 4) language difficulties (3.5%). 28.4% of the respondents were in at least one of the 'high-risk' categories for HIV infection. 26.5% were intravenous drug misusers; 15% were prostitutes; 9.8% (or their sexual partners) had lived in Central or East Africa since 1977; 5.9% were male homosexuals and 0.5% were haemophiliacs. 5.1% were infected with HIV or had Acquired Immunodeficiency Syndrome (AIDS). Only 28.8% of individuals always used condoms in short-term sexual relationships. 44.4% of respondents believed that everyone should be tested for HIV. CONCLUSION: Over one-quarter of the respondents were in higher-risk groups for infection with HIV. Almost one-third could or would not respond. It is concluded that it is not possible to identify by questionnaire, individuals at higher risk of HIV infection in forensic medical practice. This reinforces the necessity of observing good clinical practice to reduce contamination risks in this work environment. It is clear that education about risks for HIV infection is still much needed.

[Rapid recovery after colonic resection. Multimodal rehabilitation by means of Kehlet's method practiced in Vasteras]. / Nytt vårdkoncept ger snabb återhämtning efter kolonresektion. Multimodal rehabilitering enligt Kehlet praktiserad i Västerås.

UNLABELLED: By using a multimodal rehabilitation program professor Kehlet has shown accelerated recovery after colonic surgery with hospital stay of only two days, irrespective of open or laparoscopic technique. These results have not been confirmed in other studies. The aim of this study was to replace our traditional approach with Kehlet's multimodal regimen and try to reproduce his reported data. METHODS: 22 patients (median age 67 years) underwent right- or leftsided colectomies, 15 open (7 with midline incisions) and 7 laparoscopically. Continuous thoracic epidural, immediate mobilization and oral nutrition were used. Discharge was planned three days after surgery. On the first postoperative day all had oral intake and on the third day patients were mobilized for a median of 9.7 hours and all had resumed defecation. Pain and fatigue scores (VAS) were low. The median post-operative hospital stay was 3.5 (range 3-8) days. Two patients returned with complications. No cardiopulmonary or infectious complications were seen. The multimodal rehabilitation programme resulted in a quick recovery and a hospital stay of three days in most patients after colonic surgery.

Advanced biomatrix designs for regenerative therapy of periodontal tissues.

Periodontitis is an inflammatory disease that causes loss of the tooth-supporting apparatus, including periodontal ligament, cementum, and alveolar bone. A broad range of treatment options is currently available to restore the structure and function of the periodontal tissues. A regenerative approach, among others, is now considered the most promising paradigm for this purpose, harnessing the unique properties of stem cells. How to make full use of the body's innate regenerative capacity is thus a key issue. While stem cells and bioactive factors are essential components in the regenerative processes, matrices play pivotal roles in recapitulating stem cell functions and potentiating therapeutic actions of bioactive molecules. Moreover, the positions of appropriate bioactive matrices relative to the injury site may stimulate the innate regenerative stem cell populations, removing the need to deliver cells that have been manipulated outside of the body. In this topical review, we update views on advanced designs of biomatrices-including mimicking of the native extracellular matrix, providing mechanical stimulation, activating cell-driven matrices, and delivering bioactive factors in a controllable manner-which are ultimately useful for the regenerative therapy of periodontal tissues.

Biochemical changes caused by decellularization may compromise mechanical integrity of tracheal scaffolds.

Tissue-engineered airways have achieved clinical success, but concerns remain about short-term loss of biomechanical properties, necessitating a stent. This study investigated the effect of chemical-enzymatic decellularization on biochemical properties of trachea important for cell attachment and vascularization (fibronectin and laminin) and cartilage matrix homeostasis (type II collagen and glycosaminoglycans (GAG)), as well as biomechanical status. Native trachea was used as a control, and NDC trachea stored in phosphate buffered saline (PBS) in parallel to decellularization was used as a time-matched control. Decellularization removed most cells, but chondrocytes and DNA remained after 25 cycles. Fibronectin was retained throughout the lamina propria and laminin at basement membranes. DNA accumulation along ECM fibres was seen. A decline in soluble collagen was observed in decellularized tissue. GAG content of cartilage rings was reduced, even in PBS control tissue from 20 cycles onwards (p<0.05), but decellularization caused the greatest loss (p<0.01). Tensile strength declined throughout the process, but was significant only at later time points. The data demonstrate that the substantial reduction in GAG might contribute to loss of mechanical integrity of biotracheas. Overcoming structural changes that cause an imbalance in cartilage matrix equilibrium will be necessary to optimize clinical benefit, enabling widespread use of biotracheas.

'Young' oral fibroblasts are geno/phenotypically distinct.

Wound healing within the oral mucosa results in minimal scar formation compared with wounds within the skin. We have recently demonstrated distinct differences in the aging profiles of cells (oral mucosal and patient-matched skin fibroblasts) isolated from these tissues. We hypothesized that the increased replicative potential of oral mucosal fibroblasts may confer upon them preferential wound-healing capacities. Passage-matched early cultures of oral mucosal fibroblasts and skin fibroblasts demonstrated distinct gene expression profiles, with several genes linked to wound healing/tissue repair. This was related to an increased ability of the 'replicatively younger' oral mucosal fibroblasts to repopulate a wound space and reorganize their surrounding extracellular matrix environment, key activities during the wound-healing process. We conclude that oral mucosal fibroblasts exhibit a preferential healing response in vivo, due to their 'replicatively younger' phenotype when compared with that of patient-matched skin fibroblasts.

Increased oral fibroblast lifespan is telomerase-independent.

Oral mucosal wound-healing is characterized by rapid re-epithelialization and remodeling, with minimal scar formation. This may be attributed to the distinct phenotypic characteristics of the resident fibroblasts. To test this hypothesis, we investigated patient-matched oral mucosal and skin fibroblasts. Compared with skin fibroblasts, oral mucosal fibroblasts had longer proliferative lifespans, underwent more population doublings, and experienced senescence later, which was directly related to longer telomere lengths within oral mucosal fibroblasts. The presence of these longer telomeres was independent of telomerase expression, since both oral oral mucosal fibroblasts and skin fibroblasts were negative for active telomerase, as assessed according to the Telomeric Repeat Amplification Protocol. This study has demonstrated that, compared with skin fibroblasts, oral mucosal fibroblasts are 'younger', with a more embryonic/fetal-like phenotype that may provide a notable advantage for their ability to repair wounds in a scarless fashion.

The effect of mechanical strain on protease production by keratinocytes.

BACKGROUND: Intact skin is under constant tension, transmitted from the underlying dermis, but when tension is lost (i.e. upon wounding) protease activity is upregulated. OBJECTIVES: To investigate the effect of mechanical strain on protease production by both normal and transformed keratinocytes in vitro. METHODS: Keratinocytes were seeded on to membranes precoated with either type I or type IV collagen. After 48 h medium was replaced with serum-free medium and mechanical strain was applied. RESULTS: Mechanical strain resulted in decreased urokinase-type plasminogen activator (uPA) production by normal human keratinocytes (P<0.05) but increased production by transformed keratinocytes (P<0.05) cultured on type I and type IV collagen. CONCLUSIONS: Differential production of uPA by normal and transformed keratinocytes is relevant in the context of normal function, wound healing and tumorigenesis.

Minimally invasive treatment of hemiacidrin soluble staghorn renal stones.

Among 78 patients with hemiacidrin soluble staghorn renal stones, 23 were treated with a combination of one session of percutaneous debulking and high-energy extracorporeal shock wave lithotripsy (ESWL) (Group A); 17 were treated with high-energy ESWL only (Group B); and 38 were treated with low-energy only ESWL (Group C). In all patients percutaneous nephrostomies were inserted and the ESWL procedure was followed by hemiacidrin irrigation. ESWL was performed with anesthesia in Groups A and B, but without anesthesia in Group C. Groups A and B did not differ in number of ESWL sessions or length of hospital stay, and the therapeutic results at discharge and at 6-month follow-up were comparable, whereby 59% of the patients in both groups were either stone-free or had only small ( less than or equal to 3 mm) residual fragments. In Group C more ESWL sessions were performed, and the hospital stay as well as the hemiacidrin irrigation periods were longer. The fraction of stone-free renal units at discharge was significantly higher in Group C than in Group A (p less than 0.05) and after 4 weeks higher than in both Groups A (p less than 0.025) and B (p < 0.05). At 6 month follow-up, 92% of the patients in Group C were either stone-free or had only small ( less than or equal to 3 mm) residual fragments. Combined low-energy ESWL and hemiacidrin irrigation can successfully be used for treatment of even very large infection staghorn stones.

Long-term acidification of urine in patients treated for infected renal stones.

The effects of ammonium chloride, methenamine hippurate and ascorbic acid on urinary pH was studied in 14 normal subjects. A statistically significant reduction of urinary pH was recorded with ammonium chloride in daily doses of 1.5 and 3 g, but not with 2 g of methenamine hippurate or 1.8 g of ascorbic acid. Long-term treatment with ammonium chloride in doses between 1.5 and 3 g was given to 11 patients in order to reduce the risk of new stone formation or growth of fragments remaining after disintegration of infected renal stones. Biochemical stone analyses showed struvite in 9 of the treated stones, and urine cultures verified the presence of urease-producing bacteria in 10 patients. Apart from ammonium chloride, the patients were treated with antibiotics, in 4 patients continuously and in the others during periods from 2 to 34 months. The patients were followed for an average period of 32 months. No adverse reactions were recorded with the dosage used. Initially, 6 patients were stone-free, whereas 5 had residual stone fragments with a largest diameter ranging from 4 to 20 mm. At follow-up, 2 patients were still stone-free, and of 5 patients with residual fragments 1 showed stable disease and 3 an improved stone situation. In 5 patients, 3 of whom had residual stone fragments, antibiotic treatment had been interrupted without infectious relapse.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the crystallization of magnesium ammonium phosphate in urine.

The crystallization of magnesium ammonium phosphate (MAP) was studied in salt solutions and dialysed urine at similar levels of MAP supersaturation. At pH levels of 7.1 or higher crystallization occurred to the same extent in solutions with and without urinary macromolecules. Whereas crystals in the size range 3.5-5 microns were observed in the salt solution at pH 7.0, this was not so in dialysed urine. When the crystal size distribution was determined after 30 min larger crystals were observed in dialysed urine, indicating a promoting effect by urinary macromolecules on the formation of MAP crystals. A modified AP(MAP) index was formulated based on calculations with the EQUIL 2 programme in order to improve the relationship between this simplified estimate and the ion-activity product of MAP (APMAP). This index had the following form: [formula: see text] for 24-h values of magnesium (Mg), ammonium (NH4), phosphate (P), pH and urine volume (V). The APMAP required for the formation of 2,000 crystals in the size range 3.5-5 microns varied between 226 x 10(-15) and 293 x 10(-15) (mmol/l)2 in dialysed urine. An experimental system was designed based on the measurement of pH during the addition of NH4OH. At the point assumed to correspond to the start of crystallization, AP(MAP) index values between 409 and 903 were recorded. Such a test might provide useful information on the crystallization properties in urine.

An estimate of the ion-activity product of magnesium ammonium phosphate in urine.

Based on the analysis of magnesium (Mg), ammonium (NH4), phosphate (P), urine pH, and urine volume (V), a simplified estimate (AP[MAP] index) of the ion-activity product of magnesium ammonium phosphate (AP MAP) was derived: (Formula: see text). The factor A varies according to the collection period. In 4-hour urine samples more than half of the patients with staghorn calculi had values above 5 in contrast to normal subjects and calcium oxalate stone formers in whom lower values apparently were the rule. The AP(MAP) index might be of value in the evaluation and follow-up of patients with staghorn calculous disease.