O trauma em dentes decíduos é uma ocorrência comum na infância, sendo a reabsorção radicular patológica (RRP) uma das sequelas observadas no próprio dente traumatizado. A proposta deste estudo de coorte histórico foi avaliar a associação entre a ocorrência de reabsorção radicular externa e a perda precoce de incisivos centrais superiores decíduos traumatizados. Um examinador treinado e calibrado avaliou as fichas clínicas, fotografias e radiografias de prontuários de pacientes atendidos no Centro de Pesquisa e Atendimento de Traumatismos em Dentes Decíduos da FOUSP de 1998 a 2016. Após critérios de inclusão e exclusão, 521 prontuários foram analisados. Observou-se que 56,6% dos pacientes eram do sexo masculino, 54,7% tinham menos de 3 anos de idade e 30,7% tinham mordida aberta anterior e/ou sobressaliência no momento do trauma. A amostra deste estudo foi composta de 803 incisivos centrais superiores decíduos traumatizados, dos quais 69,2% sofreram trauma de tecido periodontal. Observou-se em 56 dentes (7%) reabsorção fisiológica, 138 (17,2%) reabsorção relacionada à reparação óssea (RRR), 163 (20,3%) reabsorção relacionada à infecção (RRI) e 446 (55,5%) reabsorção relacionada à expansão do folículo do sucessor permanente (RREXP). A ocorrência de reabsorção radicular patológica foi de 93% dos dentes incluídos neste estudo. A RREXP apresentou as maiores médias de tempo entre a ocorrência do trauma e o diagnóstico da reabsorção e entre o diagnóstico da reabsorção e a perda do dente, 32,3 meses e 24,1 meses, respectivamente. Já em contrapartida a RRI foi a que apresentou as menores médias 12,8 meses e 5 meses, respectivamente. A associação entre as variáveis independentes e os desfechos foram avaliadas usando análises de regressão de Poisson que permitiram o cálculo dos valores de risco relativo (RR) e respectivos intervalos de confiança de 95%. Em relação ao desfecho perda precoce, a análise de regressão de Poisson multivariada no modelo hierárquico pré-definido indicou como fatores de risco a presença de mordida aberta e/ou sobressaliência (RR = 1,95; 1,20 - 3,18), traumas de tecido periodontal do tipo luxação (RR = 1,84; 1,03 - 3,31) ou luxação com deslocamento (RR = 2,37; 1,36 - 4,12), reabsorção relacionada à reparação óssea (RR = 3,35; 1,17 - 9,55), reabsorção relacionada à infecção (RR = 7,55; 2,74 - 20,81) e a presença de necrose pulpar (RR = 2,35; 1,18 - 4,67). O tratamento endodôntico foi considerado fator de proteção (RR = 0,53; 0,36 - 0,77). Para o desfecho reabsorção desfavorável (RRR e RRI), a análise de regressão de Poisson multivariada indicou como fatores de risco trauma de tecido periodontal do tipo luxação (RR = 1,93; 1,20 - 3,09) e luxação com deslocamento (RR = 2,81; 1,80 - 4,38), traumas de tecido duro (RR = 1,94; 1,21 - 3,12) e ter coloração dental cinza ou marrom (RR = 1,46; 1,09 - 1,95). A calcificação pulpar foi considerada fator de proteção (RR = 0,51; 0,38 - 0,69). Conclui-se que a ocorrência de reabsorção radicular externa patológica em incisivos centrais superiores decíduos traumatizados é alta, e que as reabsorções relacionadas à reparação óssea e à infecção são desfavoráveis ao ciclo fisiológico destes dentes, porém o prognóstico melhora com o tratamento endodôntico. / Trauma in primary teeth is a common occurrence in childhood, and the pathological root resorption (PRR) is one of the sequelae, which may be observed in the traumatized tooth itself. The purpose of this historical cohort study was to evaluate the association between the occurrence of external root resorption and the premature loss of traumatized primary upper central incisors. One trained and calibrated examiner evaluated the dental records, photographs and radiographs of patients attended at the Research and Clinical Centre of Dental Trauma in Primary Teeth of FOUSP from 1998 to 2016. After inclusion and exclusion criteria, 521 dental records were evaluated. It was observed that 56.6% of the patients were males, 54.7% were less than 3 years of age and 30.7% had anterior open bite and/or overjet at the time of trauma. The study sample was composed of 803 traumatized primary upper central incisors, of which 69.2% suffered periodontal trauma. It was observed in 56 teeth (7%) physiological resorption, 138 teeth (17.2%) repair-related resorption (RRR), 163 teeth (20.3%) infection-related resorption (IRR) and 446 teeth (55.5%) expansion of dental sac-related resorption (EDSRR). The occurrence of pathological root resorption was 93% in the teeth included in this study. EDSRR presented the highest mean time between the occurrence of trauma and the diagnosis of resorption, and between the diagnosis of resorption and tooth loss, 32.3 months and 24.1 months, respectively. On the other hand, IRR presented the lowest averages 12.8 months and 5 months, respectively. The association between independent variables and outcomes was assessed after performance of Poisson regression analyses (regular or hierarchical model), which allowed the calculation of relative risk (RR) values and respective 95% confidence intervals. In relation to the premature tooth loss outcome, the multivariate Poisson regression analysis using the predefined hierarchical model indicated as risk factors: presence of anterior open bite and/or overjet (RR = 1.95; 1.20 - 3.18), periodontal trauma such as luxation (RR = 1.84; 1.03 - 3.31) or luxation with tooth displacement (RR = 2.37; 1.36 - 4.12), repairrelated resorption (RR = 3.35; 1.17 - 9.55), infection-related resorption (RR = 7.55; 2.74 - 20.81) and presence of pulp necrosis (RR = 2.35; 1.18 - 4.67). Endodontic treatment was considered a protection factor (RR = 0.53; 0.36 - 0.77). Regarding the desfavorable resorption outcome (RRR and IRR), the multivariate Poisson regression analysis indicated as risk factors periodontal trauma such as luxation (RR = 1.93; 1.20 - 3.09) and luxation with tooth displacement (RR = 2. 81; 1.80 - 4.38), hard tissue trauma (RR = 1.94; 1.21 - 3.12) and had grey or brown tooth colour (RR = 1.46; 1.09 -1.95). Pulp calcification was considered a protection factor (RR = 0.51; 0.38 -0.69). It is concluded that the occurrence of pathological external root resorption in traumatized primary upper central incisors is high, and repair-related and infection-related resorptions are unfavourable to the physiological cycle of these teeth, however the prognosis improves when endodontic treatment is performed.

Na gravidez, a deficiência de cálcio tem sido associada a complicações como pré-eclâmpsia, hiperparatireoidismo, osteoporose e desnutrição, o que aumenta a frequência de crianças pré-termo, de baixo peso e pequenas para a idade gestacional (PIG). Este estudo teve como objetivo avaliar o estado nutricional de cálcio em gestantes no terceiro trimestre gestacional, utilizando como parâmetros a ingestão dietética, o cálcio urinário e o telepeptídio carboxiterminal do colágeno (CTx) no plasma. Cinquenta e duas gestantes foram selecionadas no Serviço de Obstetrícia do HU-USP. As participantes responderam um registro alimentar (3 dias) e um recordatório de 24h. Para a análise dos alimentos consumidos foi utilizado o software Nutriquanti (GALANTE & COLLI, 2007). Verificou-se a distribuição normal da ingestão de cálcio e ajustaram-se os dados pela energia de acordo com o método do nutriente residual, posteriormente corrigidos pela variabilidade intrapessoal e interpessoal. Só uma gestante teve ingestão maior do que a AI (1.000 mg/d), sendo, portanto, a única que teve ingestão de Ca adequada, segundo a referência AI. Por outro lado, considerando o CTx (não aumentado em relação ao Intervalo de Referência), e considerando que 60% das gestantes apresentam hipercalciuria, supõe-se que o equilíbrio de Ca se deu pela absorção intestinal e a reabsorção renal e não pela reabsorção óssea, indicando ingestão de Ca adequada (612 ±187 mgCa/d). / During pregnancy, calcium deficiency has been associated with complications such as pre-eclampsia, hyperparathyroidism, osteoporosis and malnutrition, which increase the frequency of preterm babies, low-weighed and small for their gestational age (SGA). This study aimed to evaluate calcium nutritional status of pregnant women in the last third of pregnancy, using diet intake, urinary calcium and plasma colagen carboxy-terminus telepeptide (CTx) as parameters. Fifty-two pregnant women were selected in the Obstetrics Service of the University Hospital (HU-USP). The subjects completed a food-consumption record (3 days) e a 24-h record. For the consumed-food analysis, Nutriquanti software (GALANTE & COLLI, 2007) was used. A normal distribution of calcium intake was observed when the data were adjusted for energy according to the residual-nutrient method and corrected to consider intrapersonal and interpersonal variability afterwards. Only one pregnant woman showed a higher intake than the AI (1.000 mg/day), and therefore only this one is considered to present an adequate Ca intake when the reference is the AI. On the other hand, considering that CTx was not increased in relation to the Reference Interval and that 60% of the pregnant women presented hypercalciuria, we can say that Ca balance was due to intestinal absorption and renal resorption rather than bone resorption, thus indicating an adequate Ca intake (612 ±187 mgCa/day).

O objetivo deste estudo foi avaliar a influência da força de intrusão na ocorrência da reabsorção radicular dos incisivos superiores, de pacientes tratados com extrações de pré-molares. Para tanto, foram selecionados 56 pacientes com sobressaliência acentuada, tratados com extrações pela técnica Edgewise simplificada, sendo que metade deles apresentava concomitantemente sobremordida profunda, corrigida com arcos de nivelamento incorporando acentuação e reversão da curva de Spee, sendo este último denominado Grupo 1. Essa amostra foi obtida no arquivo da disciplina de Ortodontia da Faculdade de Odontologia de Bauru - USP, sendo que o Grupo 1 possuía 28 pacientes com sobremordida e sobressaliência acentuadas com médias de 4,78 ± 1,18 mm e 6,48 ± 2,52 mm, respectivamente, apresentando idade média de 13,41 ± 2,38 anos. O Grupo 2 contava também com 28 pacientes apenas com a sobressaliência acentuada, com média de 5,67 ± 2,73 mm, sobremordida normal, com média de 1,12 ± 0,97 mm e apresentando idade média de 13,27 ± 1,85 anos, ao início do tratamento. Utilizaram-se radiografias periapicais pré e pós-tratamento para avaliar o grau de reabsorção radicular classificando-a nos escores de Malmgren, e comparou-se entre os dois grupos pelo teste Mann-Whitney. Além disso, verificou-se a correlação entre o grau de reabsorção com os trespasses iniciais, com a quantidade de correção desses trespasses, com as quantidades de movimentação apical vertical e horizontal, obtidos pela avaliação das telerradiografias iniciais e finais, e o tempo de tratamento, utilizando o coeficiente de correlação de Spearman. Também uma comparação entre os incisivos centrais e laterais foi executada, em ambos os grupos, para definir qual foi mais afetado, utilizando-se o teste de Wilcoxon. Os resultados revelaram maior reabsorção no grupo tratado com mecânica intrusiva, uma correlação estatisticamente significante da reabsorção com a quantidade de sobremordida inicial e com seu grau de correção, e diferente grau de reabsorção entre os incisivos apenas no grupo 2, onde os incisivos laterais apresentaram-se mais afetados, porém com diferença estatística não significante. / The objective of this study was to evaluate the influence of intrusion force in the occurrence of root resorption in the maxillary incisors, in 56 patients treated with premolars extraction in the Edgewise technique. The sample was retrospectively selected from the files of the Orthodontic Department at Bauru Dental School, University of São Paulo. Group 1 comprised 28 patients with accentuated overbite and overjet (4.78 ± 1.18 mm and 6.48 ± 2.52 mm, respectively). Group 2 also was composed by 28 patients and presented accentuated overjet (5.67 ± 2.73 mm), but with normal overbite (1.12 ± 0.97 mm). The initial mean ages were 13.41 ± 2.38 and 13.27 ± 1.85 years, respectively. Periapical radiographs from the pre and post treatment period were used to evaluate the amount of root resorption, according to the method of Malmgren. The groups were compared with the nonparametric Mann-Whitney test. The correlation between the degree of root resorption with the initial overjet, the amount of overjet correction, the amount of vertical and horizontal movement of the root apex, obtained in the cephalograms, and the treatment time were evaluated with the Spearman correlation coefficient. A comparison of the degree of root resorption between the central and lateral incisors, to define which was the most affected, was performed with the Wilcoxon test. The results showed a greater amount of root resorption in the group of patients treated with both retraction and intrusion mechanics. The amount of root resorption was statistically significant correlated with the initial overbite and with the amount of its correction. The maxillary central and lateral incisors were similarly affected in the amount of root resorption.

Objectives: Tooth extraction initiates a cascade of events that often leads to local anatomic changes in the alveolar ridge. Ridge preservation is a surgical approach aimed at minimizing hard and soft tissue volume loss. There have been contradicting reports on the efficacy of socket grafting for alveolar ridge preservation. Interestingly, there is a paucity of adequately powered randomized controlled clinical trials. The purpose of this study was to assess the effect of the application of a socket grafting technique on alveolar ridge dimensional changes following tooth extraction. Methods: Healthy patients requiring the extraction of one single-rooted tooth on either arch, from second premolar to second premolar, excluding mandibular incisors, and who met the eligibility criteria were recruited. Patients were then randomly assigned to either the control group, consisting of tooth extraction alone, or the experimental group, which consisted of extraction and simultaneous ridge preservation using an allograft bone material to fill the socket and a dense polytetrafluoroethylene membrane (dPTFE) to seal it. Cone beam computed tomography (CBCT) was obtained immediately prior to extraction (baseline) and at 14 weeks. Linear measurements with the use of a tooth-supported stent were obtained immediately after extraction (baseline) and at 14 weeks. Linear and volumetric measurements were made using data obtained from the CBCTs. Masked, calibrated examiners performed all radiographic measurements. Measurements obtained included buccal keratinized tissue width, buccal and lingual plate height and width, alveolar ridge horizontal width (CBCT); and alveolar ridge volume changes. Digital planning of dental implants was performed in the ideal restorative location and need for additional grafting was virtually determined. The primary outcome of interest was volumetric reduction of the alveolar ridge at 14 weeks. Linear mixed model statistical analyses were used to compare the mean change in the measurements between the grafted and control groups. Results: A total of 59 subjects were recruited, of which 53 patients (27 control and 26 experimental) completed the study. No statistically significant difference was found between the two groups at baseline for any of the parameters analyzed. At the 14 week follow-up appointment there was an average loss in height of the buccal plate of 1.17 mm and 0.61 mm for the control (CG) and experimental (ARP) groups, respectively, showing statistical significance (p=0.012). The lingual plate height was reduced 0.7 mm in CG and 0.47 mm in ARP with no statistical significance (0.075). A linear loss in the buccal-lingual dimension of the alveolar ridge was noted radiographically in both groups, 1.68mm in CG and 1.07mm in ARP, which demonstrated a statistical significant difference between them (p=0.023). Volumetric analysis demonstrated a mean volume loss of 15.83% in the CG showing statistical significance from the 8.36% loss shown in the ARP group. This difference demonstrates a clinical significance when virtual planning of implant placement in the ideal restorative location revealed the need for additional grafting at 13/27 or 48% of CG and 3/26 or 11% of ARP sites. Additionally, a very robust, statistically significant correlation was noted between buccal bone plate width and reduction of alveolar bone volume after 14 weeks of healing (p< 0.0001). A multivariate regression analysis revealed that within the control group a buccal plate <1mm lead to >10% volumetric reduction, while the same reduction in the graft group was only seen when the buccal plate was less that 0.6mm. Conclusions: In this study, a novel volumetric analysis of alveolar ridge reduction after tooth extraction was performed, which demonstrated that socket grafting for alveolar ridge preservation does provide a therapeutic benefit. This finding was associated to a decreased probability of requiring additional grafting at the implant site. The thickness of the buccal plate at the time of extraction appears to be a valuable factor to predict the amount of resorption that will take place, meaning that more resorption should be expected, as the buccal plate gets progressively thinner.

[Truncated abstract] Osteoclasts are multinucleated giant cells responsible for the resorption of the mineralized bone matrix during the process of bone remodelling. During activation towards bone resorption, polarization of the osteoclast results in the formation of a unique plasma membrane, the ruffled border, the actual resorptive organelle of the osteoclast. Through this domain protons are actively pumped into the resorption lacuna creating an acidic microenvironment that favours the dissolution of the mineralized bone matrix. The polarised secretion of protons is carried out by the action of the vacuolar-type (H+)-ATPase (V-ATPase), composed of functionally and structurally distinct subunits of the V1 and V0 domains. The general structure of the V-ATPase complex is highly conserved from yeast to mammals, however, multiple isoforms for specific V-ATPase subunits do exist exhibiting differential subcellular, cellular and tissue-specific localizations. This study focuses on the molecular identification and characterization of V-ATPase accessory subunit Ac45 and the d2 isoform of the V0 domain d subunit in osteoclasts. Using the techniques of cDNA Subtractive Hybridization and DNA Micro-Array analyses respectively, the accessory subunit Ac45 and the d2 isoform of the V0 domain d subunit were identified in RAW264.7-cells derived OcLs. ... Using web-based computational predictions, two possible transmembrane domains, an N-terminus 'signal anchor' sequence and a C-terminus dilysine- like endoplasmic reticulum (ER) retention signal were identified. By confocal microscopy, EYFP-tagged e was found to localize to the perinuclear region of transfected COS-7 cells in compartments representing the ER and Golgi apparatus with some localization in late endosomal/lysosomal-like vesicles. ER truncation of e did not alter its subcellular localization but exhibited significantly weaker association with Ac45 compared to the wild-type as depicted by BRET analyses. Association with the other V0 subunits remain unaffected. This may hint at a possibility that Ac45 may play a role in the masking of the ER signal of e following it's incorporation into the V0 domain. Although no solid evidence for a role in the assembly of the mammalian VATPase have been established, subunit e still represents a potential candidate whose role in the V-ATPase complex requires further investigation. Collectively, the data presented in this thesis has provided further insight into the composition of the osteoclast V-ATPase proton pump by: 1) identifying an accessory subunit, Ac45 which shows promise as a potential candidate for the regulation and/or targeting of the V-ATPase complex in osteoclasts and truncation of its targeting signal impairs osteoclastic bone resorption; 2) identification and preliminary characterization of the d2 isoform of the V0 domain d subunit whose exact role in the V-ATPase complex and in osteoclasts remains to be determined, although its has been implicated to be essential for osteoclastic function; and 3) Preliminary characterization of subunit-e, a potential assembly factor candidate for the mammalian V-ATPase V0 domain.

<p>The purpose of this study was to investigate the various types of onsite greywater treatment facilities available at two housing communities (Hull Street and Moshoeshoe Eco Village) in Kimberley, South Africa. The objective was to undertake a close observation through personal experience of the installations, measure water consumption and greywater produced, do an inventory of household cleaning chemicals and conduct interviews of different stake-holders of the Housing Project to find out their views on greywater and Ecosan issues. The study was conducted between June and August 2006.</p><p>The average water consumption per household per day during the study period was 272 L and 170 L in Eco Village and Hull Street respectively. The average greywater produced per household per day was 190 L and 119 L in Eco Village and Hull Street respectively. In Hull Street, the average water consumed and greywater produced per person per day during this study was 51L and 36L respectively. Three main types of treatment systems were installed in the study area; sandfilters, infiltration pits and resorption trenches. The sandfilters were poorly designed and were not functioning properly. The infiltration pits though working they were experiencing problems of poor infiltration and required constant draining and maintenance in many homes, especially those that have high water consumption and produce much greywater. The resorption trenches that make use of aerobic mulch media followed by infiltration had been installed in one house unit and after about 7 months had not presented problems to the user. Close monitoring done on this facility for about 4 weeks showed proper functioning according to its design.</p><p>Quite a lot had been done over time to improve on the installations in Hull Street and Eco village. The toilet installations have been exchanged and a number of alternatives to improve on the treated greywater have been attempted. The users and the housing company’s personnel feel one of the major problems being encountered is in treating greywater. Appropriate ways to compost faecal matter are still being sought. Hence use of greywater, urine and composted faeces in urban agriculture by residents is yet to be visible and will need encouragement.</p><p>Generally, the residents at Hull Street and Eco Village like the community life, house structures and location. However, they wish that improvement be made in some areas to make life in these areas more comfortable. The residents of both Hull Street and Eco Village expect better greywater treatment facilities. The community in Hull Street requests shopping centres, sport facilities, fence around the area, and taxi services among others. It is important to note that many people did not ask for further improvements on the toilet systems which might indicate they are coping with the urine diversion alternative sanitation.</p><p>The user perception on whole was good, but the need for constant attention and maintenance seems to offer a hurdle to the infiltration and sand filter facilities to treat greywater.</p>

<p>Fibroblasts continuously strive to organize and compact the surrounding extracellular matrix (ECM). Recent data suggest that this cellular contractility controls interstitial fluid homeostasis in loose connective tissues (CT). The aim of this thesis was to study the effects of prostaglandins on fibroblast contractility and to investigate whether fibroblasts in the interstitial CT surrounding the human endolymphatic duct (ED) can modulate inner ear fluid pressure and endolymph resorption. </p><p>Paper I shows that prostaglandin E1 (PGE₁) and prostacyclin inhibit fibroblast-mediated collagen matrix compaction <i>in vitro</i> and lower the interstitial fluid pressure <i>in vivo</i> in rat dermis. Paper II demonstrates that the inhibition of collagen matrix compaction by PGE₁ is protein kinase A-dependent. Furthermore, PGE₁ induces a complete but reversible actin depolymerization in human dermal fibroblasts by affecting the phosphorylation state of regulatory actin-binding proteins. Paper III describes that the cells of the interstitial CT encompassing the human ED are organized in a network based on intercellular- and cell-ECM contacts. Paper IV shows that two distinct cell phenotypes populate this interstitial CT: one expressing the lymph endothelial marker podoplanin and the other a fibroblast marker. Furthermore, CT cells isolated from human ED tissues exhibited the same tissue compacting properties <i>in vitro</i> as dermal fibroblasts. </p><p>In conclusion, PGE₁ inhibits fibroblast contractility by interfering with the stability and dynamics of the actin cytoskeleton, which leads to a loss of integrin-mediated adhesion to the ECM. These mechanisms are supposedly involved in edema formation in skin during inflammation and might be involved in the formation of endolymphatic hydrops in the inner ear of patients with Ménière’s disease.</p>

The calcitonin gene family of peptides consists of calcitonin (CT), two calcitonin gene related peptides (α-CGRP, β-CGRP), adrenomedullin (ADM), amylin (AMY), three calcitonin receptor activating peptides (CRSP1-3) and intermedin/adrenomedullin2 (IMD). These peptides bind to one of two G protein -coupled receptors, the calcitonin receptor (CTR) or the calcitonin receptor-like receptor (CRLR). The receptor specificity to different ligands is dependent on the formation of a complex with one of three receptor activity-modifying proteins (RAMP1-3). The aim of this study was to analyse effects of this family of peptides on the formation of osteoclasts and bone resorption, and the expression of the receptor components in bone cells. CT inhibited the formation of multinucleated osteoclasts in spleen cell cultures and in bone marrow macrophage cultures (BMM) without affecting a number of genes important for osteoclast differentiation, activity or fusion of osteoclast progenitor cells. All members of the CT family, except ADM, inhibited osteoclastogenesis in BMM. The inhibitory effect seemed to involve activation of both protein kinase A and the exchange protein directly activated by cyclic AMP (Epac) signalling. BMM expressed the CRLR, RAMP1-3 and the receptor component protein (RCP). AMY, ADM, CGRP and IMD, but not CRSP and CT, increased cyclic AMP (cAMP) levels in these cells, indicating the presence of functional receptors. Stimulation of BMM with RANKL gradually increased the levels of CTR mRNA as well as the capacity of the cells to respond to the stimulation by CRSP and CT. The response to stimulation of ADM was, on the contrary, decreased by RANKL. Stimulation of RANKL caused a transiently enhanced CRLR mRNA expression and transiently decreased RAMP1, but did not affect RAMP2, RAMP3, or RCP mRNA. However, RANKL did not affect protein levels of CRLR or RAMP1-3. CT, CGRP, AMY, ADM, IMD and CRSP all down regulated the CTR mRNA, but none of the peptides caused any effects on the expression of CRLR or any of the RAMPs. All members of the CT family, except ADM, rapidly and transiently, inhibited bone resorption in mouse calvarial bones. CT, CGRP, AMY and CRSP also significantly stimulated cAMP formation in the calvaria. cAMP analogues specifically stimulating the PKA or the Epac pathways did not cause inhibition of bone resorption in the calvaria. An unspecific cAMP analogue, stimulating both pathways did, however, cause inhibition. Analyses of an osteoblastic cell line, MC3T3-E1, showed that these cells express the mRNA for CRLR and all three RAMP proteins. In conclusion, the results of this thesis show that all peptides in CT family of peptides, except ADM, inhibit of bone resorption and osteoclast formation and that these effects involve the adenylate cyclase-cAMP pathway. Furthermore, expressions of CRLR and RAMP1-3 mRNA have been demonstrated on osteoclasts, as well as in an osteoblastic cell line.

Bone metabolism is regulated by endocrine and paracrine signalling molecules influencing bone cells in the continuously remodelling bone tissue. These molecules include a variety of osteotropic stimulatory and inhibitory cytokines. Degradation of alveolar bone in periodontal disease is believed to be a result of local release of such osteotropic cytokines, although the relative importance of particular cytokines and their cellular origin is currently unknown. The aim of the present project was to study if, and how, pro-inflammatory cytokines in the interleukin-6 (IL-6) family of cytokines, and anti-inflammatory IL-4 and IL-13 type of cytokines, can affect osteoclast differentiation and bone resorption. Additionally, the objective was to study if gingival fibroblasts may influence alveolar bone resorption through secretion of IL-6 type cytokine release and if the secretion is regulated by pro-inflammatory as well as anti-inflammatory mediators such as IL-4 and IL-13. IL-6 in combination with its soluble receptor (sIL-6R) was found to stimulate mouse calvarial bone resorption. Similarly, two other IL-6 family members, leukemia inhibitory factor (LIF) and oncostatin M (OSM) were found to stimulate bone resorption. The stimulatory effect on bone resorption induced by the three cytokines was associated with increased expression of receptor activator of NF- κB ligand (RANKL), a cytokine which is essential in osteoclast formation and activation through binding to receptor activator of NF- κB (RANK) on osteoclastic cells. The interaction between RANKL and RANK can be inhibited by binding of the decoy receptor osteoprotegerin (OPG) to RANKL, and the expression of OPG was also regulated by IL-6, LIF and OSM (Paper I). The two related cytokines IL-4 and IL-13 were found to inhibit osteoclastogenesis and mouse calvarial bone resorption by mechanisms involving a decreased RANKL/OPG ratio in osteoblasts and decreased RANK expression in osteoclastic cells. The results further demonstrated that IL-4 and IL-13 exert their effects on both osteoblasts and osteoclasts by a mechanism involving the transcription factor signal transducer and activator of transcription 6 (STAT6) (Paper II). Constitutional expression of IL-6, LIF and another member of the IL-6 family of cytokines, IL-11, was demonstrated in human gingival fibroblasts. IL-6 type cytokine expression levels were found to be enhanced by IL-1β and tumour necrosis factor-α (TNF-α) (Paper III), whereas IL-4 and IL-13 inhibited IL 11 and LIF release from gingival fibroblasts (Paper IV). In conclusion, IL 6 type cytokines were found to be stimulators and IL-4 and IL-13 inhibitors of bone resorption in vitro via mechanisms involving RANK/RANKL/OPG interactions. Additionally, gingival fibroblasts were able to secrete several cytokines in the IL-6 family. Secretion was further enhanced by pro-inflammatory mediators and inhibited by IL-4 and IL- 13. These findings support the view that resident cells may influence the pathogenesis of periodontal disease through osteotropic cytokine production.

Bone tissue is continuously degraded and rebuilt to respond to the needs of the body. Cells of the osteoblast lineage are responsible for the formation of bone, whereas the resorption of bone tissue is carried out by osteoclasts. To prevent imbalance between bone formation and resorption, these processes are delicately regulated by a complex network of both systemic factors and factors produced locally in the bone microenvironment, including members of the IL-6 family of cytokines. During the last decades, the presence of nerve fibers in skeletal tissue and presence of receptors for several neurotransmitters on both osteoblasts and osteoclasts, have suggested a possible role for neuropeptides in the regulation of skeletal metabolism. The overall aim of this study was to investigate the roles of cytokines in the IL-6 family and the neuropeptide VIP in regulation of osteotropic cytokine expression and bone metabolism in vitro. In Paper I, stimulation of bone resorption by the cytokine IL-6, in the presence of its soluble receptor sIL-6R, was demonstrated in mouse calvarial bones. OSM and LIF, other members of the IL-6 family of cytokines, were also shown to increase bone resorption. Furthermore, IL-6+sIL-6R, LIF, and OSM increased the expression of RANKL, which by binding to its receptor RANK functions as a crucial inducer of osteoclast formation and activation. In Paper II-IV, the effects of the neuropeptide VIP and related peptides on expression of osteotropic cytokines by osteoblasts and bone resorption in vitro have been studied. VIP and PACAP-38 both increased IL-6 production in osteoblasts in a time- and concentration-dependent manner. In contrast, no effect was seen with the related peptide secretin, indicating that the effects were mediated by the VPAC2 receptor. VIP and PACAP, in contrast to secretin, also induced IL-6 promoter activity in osteoblastic MC3T3-E1 cells transfected with an IL-6 promoter/luciferase construct. The effects of VIP on IL-6 were shown to be mediated by several intracellular pathways, including cAMP/PKA/CREB, AP-1, and C/EBP, but not NF-kB or the cAMP-activated Epac pathway. The release of IL-6 from osteoblasts was increased by several pro-inflammatory osteotropic cytokines, including interleukin-1b, an effect that was further potentiated by VIP, indicating a possible neuro-immunomodulatory interaction in the regulation of bone metabolism. VIP and PACAP-38 also increased the osteoblastic expression of RANKL and decreased the expression of OPG and M-CSF, factors crucial in regulation of differentiation and activation of osteoclasts. Although this indicated a possible bone resorptive effect, VIP was found to decrease osteoclast formation and bone resorption by directly targeting osteoclast progenitor cells through an inhibitory mechanism. In conclusion, the results in this thesis indicate that several cytokines in the IL-6 family stimulate bone resorption in calvarial bones in vitro, most likely through the RANKL-RANK interaction. Furthermore, expression of the osteotropic cytokine IL-6 in osteoblasts is stimulated by the neuropeptide VIP through VPAC2 receptors via several intracellular pathways, further strengthening the role of neuropeptides as local regulators of bone metabolism.