Revitalisation endodontic treatment of traumatised immature teeth: a prospective long-term clinical study.

PURPOSE: Continuation of root development following revitalisation endodontics (RET) has been shown to be unpredictable with lower success rates in traumatised teeth. This study reports the outcomes for RET in traumatised teeth over a review period of 4 years. METHODS: A prospective uncontrolled study, where RET was performed on traumatised upper immature anterior teeth with necrotic pulps in 15 children (mean age = 8.3 years), was conducted. Patients were reviewed at 3, 9, 12, 24, and 48 months, where clinical and radiographic assessments were performed. At the last review appointment, patients and parents answered questions assessing their perception and acceptance of tooth colour change over time. McNemar's Exact test and linear mixed model assessment were used to assess changes in pulpal electrical response and radiographic evidence of continuation of root development over time, respectively. RESULTS: There was 83.3% healing with no significant changes in EPT responses, and no significant changes in root lengths, while significant changes in root widths (p < 0.05) and root apex widths (p < 0.001) were found over time. Twenty-five percent of patients and 33% of parents felt that there were changes in tooth colour following RET over time. CONCLUSION: Within the limitations of this study, traumatised teeth treated using RET showed no significant root lengthening, however, acceptable periapical healing, slow thickening of root dentinal walls, and rapid development of apical closure were evident over a period of 43 months. Using Portland cement and omitting minocycline, did not eliminate crown colour change following RET.

A review of the effect of music on dental anxiety in children.

AIM: To review the current literature on the effectiveness of using music as an intervention to reduce dental anxiety in children. METHODS: At the University of Leeds, the School of Music and the School of Dentistry collaborated to conduct an online search strategy. The Cochrane Library and Medline databases were used to find the current available evidence. RESULTS: Systematic reviews and clinical trial studies as well as cohort studies containing pertinent information on the effect of music on anxiety in the clinical setting were reviewed. The literature showed that music can have a biological and psychological impact on emotion and consequently has been used effectively as an aid to moderate anxiety in the clinical setting. With regard to paediatric dentistry, majority of studies were found to support the use of music in reducing dental anxiety in children, however several additional studies showed that music did not significantly reduce the children's dental anxiety. The studies employed a number of methods to measure dental anxiety including the Venham's Picture Test, the Venham's clinical anxiety rating scale and pulse oximetry. They also used a range of music types; some studies allowed for patient self-selection of music whereas others dictated the music the children listened to. CONCLUSIONS: There is an increasing body of evidence to support the use of music to moderate anxiety within the clinical setting in both medicine and dentistry. However, the current evidence for the effectiveness of using music to reduce dental anxiety in children is inconclusive and of limited quality.

Mantle cell lymphoma: primary oral presentation.

Mantle-cell lymphoma is an uncommon lymphoid malignancy of B-cells. It is often aggressive and prognosis is poor. A 69-year-old gentleman with a history of ischaemic heart disease was referred from primary care with a painless right floor of mouth swelling that had been present for 1 month. He otherwise completely asymptomatic. Incisional biopsy of the lesion was undertaken and marker studies demonstrated mantle cell lymphoma. Positron emission tomography-computed tomography and bone marrow biopsy showed widespread but low volume involvement. The patient was referred to the haematology multidisciplinary team for further assessment and treatment.

Human corneal epithelial equivalents constructed on Bombyx mori silk fibroin membranes.

Membranes prepared from a protein, fibroin, isolated from domesticated silkworm (Bombyx mori) silk, support the cultivation of human limbal epithelial (HLE) cells and thus display significant potential as biomaterials for ocular surface reconstruction. We presently extend this promising avenue of research by directly comparing the attachment, morphology and phenotype of primary HLE cell cultures grown on fibroin to that observed on donor amniotic membrane (AM), the current clinical standard substrate for HLE transplantation. Fibroin membranes measuring 6.3 ± 0.5 µm (mean ± sd) in thickness and permeable to FITC dextran of a molecular weight up to 70 kDa, were used. Attachment of HLE cells to fibroin was similar to that supported by tissue culture plastic but approximately 6-fold less than that observed on AM. Nevertheless, epithelia constructed from HLE on fibroin maintained evidence of corneal phenotype (K3/K12 expression) and displayed a comparable number and distribution of ΔNp63(+) progenitor cells to that seen in cultures grown on AM. These results support the suitability of membranes constructed from Bombyx mori silk fibroin as substrata for HLE cultivation and encourage progression to studies of efficacy in preclinical models.

Effects of fibroblast origin and phenotype on the proliferative potential of limbal epithelial progenitor cells.

The current study investigates potential differences in fibroblast phenotype across the anterior segment of the human eye with the aim to understanding factors that support the regenerative function of human limbal epithelial progenitor cells (LEPs) during wound healing. Separate cultures of fibroblasts were established from the cornea, limbus and sclera by growth in serum-supplemented medium. The resulting cultures were examined for potential differences in morphology and growth rate, as well as expression of CD34, CD45, CD90, CD141, CD271, vimentin and α-smooth muscle actin (α-sma). Finally, cultures were examined for their ability to support the growth of LEPs. While all cultures grew at a similar rate, scleral cultures often contained larger and more irregularly shaped cells which stained positive for α-sma. Western blotting confirmed a gradient of α-sma expression with lowest levels in corneal cultures. All three cultures stained positively for CD90 and vimentin, and were negative for CD34, CD45, CD141 and CD271. Only limbal or corneal irradiated fibroblasts supported the establishment of LEP cultures. While LEP colony forming efficiency and prominent expression of ABCG2, C/EBPδ and p63 was similar with either limbal or corneal fibroblasts, limbal fibroblasts supported significantly better growth. These results indicate that scleral fibroblasts have an increased capacity for myofibroblast formation which appears to negatively impact on their ability to support LEP growth. Superior growth of LEPs in the presence of limbal fibroblasts indicates a role for limbal fibroblasts in promoting the proliferation of limbal epithelium during wound healing.

Discovery and characterization of IGFBP-mediated endocytosis in the human retinal pigment epithelial cell line ARPE-19.

Insulin-like growth factor binding proteins (IGFBPs) are prime regulators of IGF-action in numerous cell types including the retinal pigment epithelium (RPE). The RPE performs several functions essential for vision, including growth factor secretion and waste removal via a phagocytic process mediated in part by vitronectin (Vn). In the course of studying the effects of IGFBPs on IGF-mediated VEGF secretion and Vn-mediated phagocytosis in the RPE cell line ARPE-19, we have discovered that these cells avidly ingest synthetic microspheres (2.0 mum diameter) coated with IGFBPs. Given the novelty of this finding and the established role for endocytosis in mediating IGFBP actions in other cell types, we have explored the potential role of candidate cell surface receptors. Moreover, we have examined the role of key IGFBP structural motifs, by comparing responses to three members of the IGFBP family (IGFBP-3, IGFBP-4 and IGFBP-5) which display overlapping variations in primary structure and glycosylation status. Coating of microspheres (FluoSpheres((R)), sulfate modified polystyrene filled with a fluorophore) was conducted at 37 degrees C for 1 h using 20 microg/mL of test protein, followed by extensive washing. Binding of proteins was confirmed using a microBCA assay. The negative control consisted of microspheres treated with 0.1% bovine serum albumin (BSA), and all test samples were post-treated with BSA in an effort to coat any remaining free protein binding sites, which might otherwise encourage non-specific interactions with the cell surface. Serum-starved cultures of ARPE-19 cells were incubated with microspheres for 24 h, using a ratio of approximately 100 microspheres per cell. Uptake of microspheres was quantified using a fluorometer and was confirmed visually by confocal fluorescence microscopy. The ARPE-19 cells displayed little affinity for BSA-treated microspheres, but avidly ingested large quantities of those pre-treated with Vn (ANOVA; p < 0.001). Strong responses were also observed towards recombinant formulations of non-glycosylated IGFBP-3, glycosylated IGFBP-3 and glycosylated IGFBP-5 (all p < 0.001), while glycosylated IGFBP-4 induced a relatively minor response (p < 0.05). The response to IGFBP-3 was unaffected in the presence of excess soluble IGFBP-3, IGF-I or Vn. Likewise, soluble IGFBP-3 did not induce uptake of BSA-treated microspheres. Antibodies to either the transferrin receptor or type 1 IGF-receptor displayed slight inhibitory effects on responses to IGFBPs and Vn. Heparin abolished responses to Vn, IGFBP-5 and non-glycosylated IGFBP-3, but only partially inhibited the response to glycosylated IGFBP-3. Our results demonstrate for the first time IGFBP-mediated endocytosis in ARPE-19 cells and suggest roles for the IGFBP-heparin-binding domain and glycosylation status. These findings have important implications for understanding the mechanisms of IGFBP actions on the RPE, and in particular suggest a role for IGFBP-endocytosis.

Vitronectin supports migratory responses of corneal epithelial cells to substrate bound IGF-I and HGF, and facilitates serum-free cultivation.

Vitronectin (VN) is a multi-functional glycoprotein best known for its effects on cell attachment and spreading, but has more recently been shown to mediate cellular responses to growth factors. The presence of VN within the tear film and expression of required receptors (alpha v integrins) on corneal epithelial cells suggests the potential for a similar role within the ocular surface. Thus we have studied the ability of VN to alter the metabolic (MTT assay) and migratory (trans-membrane migration) responses of corneal epithelial cells to growth factors associated with the ocular surface including epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and insulin-like growth factor-I (IGF-I). Our hypothesis was that culture surfaces coated with VN might selectively facilitate responses to growth factors which are known to bind VN including EGF, IGF-I (via IGF binding protein) and HGF. Metabolic responses were observed towards each growth factor when applied to the culture medium, but not towards culture plastic pre-treated with VN and, or growth factors. Optimal metabolic responses were observed towards IGF-I applied in conjunction with EGF. Migration through porous polycarbonate membrane was significantly increased when the substrate had been pre-coated with VN and IGF-I (applied in conjunction with IGFBP-3) or VN and HGF. This finding is consistent with the ability of IGF-I (via an IGFBP) and HGF to form complexes with VN and suggests that integrin/growth factor receptor co-activation is required for corneal epithelial cell migration. In further studies, VN applied in conjunction with IGF-I, IGFBP-3 and EGF (both to the culture plastic and in the culture medium) was found to support the establishment and serial propagation of limbal-corneal epithelial cell cultures in the absence of serum, but irradiated 3T3 cells (i3T3) were still necessary for culture expansion. Immunocytochemistry of resulting cultures for keratin 3 and p63 revealed a similar phenotype to those established under current best-practice conditions (i3T3, foetal bovine serum, EGF and insulin). In conclusion, our novel findings suggest a role for VN-growth factor complexes in stimulating corneal epithelial migration within the provisional wound bed and demonstrate that VN-growth factors interactions can be exploited to enable manufacture of bioengineered ocular surface tissue under serum-free conditions.

Analysis of p63 and cytokeratin expression in a cultivated limbal autograft used in the treatment of limbal stem cell deficiency.

AIM: To investigate the expression of p63 and cytokeratins throughout the course of producing a cultivated autograft of limbal epithelial cells. METHODS: A 75 year old male with a severe alkali burn to his right eye received two cultivated autografts of limbal epithelial cells on amniotic membrane followed by a corneal allograft. Immunostaining for p63 and cytokeratins was performed during ex vivo expansion with 3T3 fibroblasts, following subcultivation on amniotic membrane, and on the excised corneal button. RESULTS: Cultures grown in the presence of 3T3 fibroblasts or on amniotic membrane displayed positive staining for keratins 14 and 19, and p63, but poor staining for keratin 3 (K3). The excised corneal button possessed a stratified epithelium of K3 positive cells residing on amniotic membrane. CONCLUSIONS: Our results document for the first time the co-expression of cytokeratins 14 and 19 with p63 in a cultivated limbal graft. These data support the conclusion that cultivated grafts of limbal epithelium contain predominantly undifferentiated cells with the potential to regenerate a normal corneal epithelium.

Infrequency of pulmonary microbial colonisation prior to respiratory disease in HIV-infected individuals.

To determine whether organisms are present in the HIV-infected lung prior to clinical respiratory disease, a cross-sectional bronchoscopic comparative analysis of 39 asymptomatic HIV-positive subjects and 31 healthy controls with 2-year prospective bronchoscopic monitoring of the HIV study group was performed. Pathological examination of bronchoalveolar lavage (BAL) fluid using standard microbiological techniques was undertaken. Organisms were recovered from similar numbers of HIV-positive and control subjects (7 of 39 and 3 of 31) and comprised predominantly scanty growths of bacteria. Five subjects developed respiratory disease during follow-up. Repeat BAL was performed in 11 asymptomatic HIV-positive patients; no relationship was found between the organisms isolated at the two procedures. The findings suggest that the asymptomatic HIV-positive lung is not a frequent site of either microbial colonisation or subclinical infection. This has implications for the understanding of the pathogenesis of HIV-related pulmonary disease.

An evaluation of the cost-effectiveness of using CHROMagar for yeast identification in a routine microbiology laboratory.

CHROMagar, a chromogenic differential culture medium, is claimed to facilitate the isolation and presumptive identification of certain clinically important yeast species, e.g., Candida albicans. This study evaluated the cost-effectiveness and time advantage of using it in comparison with Sabouraud dextrose agar (SDA). Three possible pathways, each of which included the use of one or both media, were compared in a routine laboratory. A total of 21 yeast isolates was cultured from 298 clinical samples from neutropenic and AIDS patients. An overall sensitivity of 95.2% was observed for each medium and primary isolation on CHROMagar was found to be 100% sensitive and 100% specific for C. albicans. For identification purposes, after initial culture the use of CHROMagar provided the most economical and least time-consuming method. Direct inoculation on to CHROMagar is recommended for blood cultures when yeast cells are seen on microscopy and where early appropriate therapy is imperative.

Phosphorylcholine-containing antigens in bacteria from the mouth and respiratory tract.

Phosphorylcholine (PC)-containing antigens were sought in 269 bacterial isolates from the mouth and respiratory tract by an enzyme immunoassay method. Only 41 (15%) isolates were PC-positive and of these 29 (70%) were strains of Haemophilus influenzae. Other species that produced positive results included two of five isolates of Gemella haemolysans, two of five isolates of Micrococcus spp., and a single strain each of Bacillus sp., Corynebacterium jeikeium, Lactococcus sp. and H. parainfluenzae. The presence of PC-containing antigens in H. influenzae may be an important source of cross-reaction in antigen detection techniques that detect the C-polysaccharide antigen of Streptococcus pneumoniae in respiratory specimens and would result in false positive results.