The mechanisms underlying the effect of alterations in Type I collagen on bone mechanical properties are not well defined. Clinical tools for evaluating fracture risk, such as dual energy x-ray absorptiometry (DXA) and quantitative ultrasound (QUS) focus on bone mineral and cannot detect changes in the collagen matrix. The mechanical response tissue analyzer (MRTA) is a potential tool for evaluating fracture risk. Thus, the focus of this work was to investigate the effects of collagen degradation on bone mechanical properties and examine whether clinical tools can detect these changes. Female and male emu tibiae were endocortically treated with 1 M potassium hydroxide (KOH) solution for 1-14 days and then either mechanically tested in three-point bending, fatigued to failure or fatigued to induce stiffness loss. Computed Tomography scans, DXA, QUS, MRTA and three-point bend testing in the elastic region were performed on emu tibiae before and after either KOH treatment or fatigue to induce stiffness loss. Fracture surfaces were examined to determine failure mechanisms. Bone mineral and bone collagen were characterized using appropriate techniques. Bone mineral-collagen interface was investigated using Raman spectroscopy and atomic force microscopy (AFM). Endocortical KOH treatment does not affect bone mineral however, it causes in situ collagen degradation, rather than removal and may be weakening the mineral-collagen interface. These changes result in significantly compromised mechanical properties. Emu tibiae show significant decreases in failure stress and increased failure strain and toughness, with increasing KOH treatment time. The significant increase in toughness of KOH treated bones is due to structural alterations that enhance the ability of the microstructure to dissipate energy during the failure process, thereby slowing crack propagation, as shown by fracture surface analysis. KOH treated samples exhibit a lower fatigue resistance compared to untreated samples at high stresses only for both sexes. Partial fatigue testing results in similar decreases in modulus for all groups and sexes. The MRTA detected these changes whereas DXA and QUS did not. MRTA detects changes in bone mechanical properties induced by changes in collagen quality and fatigue and could be a more effective tool for predicting fracture risk.

The mechanisms underlying the effect of alterations in Type I collagen on bone mechanical properties are not well defined. Clinical tools for evaluating fracture risk, such as dual energy x-ray absorptiometry (DXA) and quantitative ultrasound (QUS) focus on bone mineral and cannot detect changes in the collagen matrix. The mechanical response tissue analyzer (MRTA) is a potential tool for evaluating fracture risk. Thus, the focus of this work was to investigate the effects of collagen degradation on bone mechanical properties and examine whether clinical tools can detect these changes. Female and male emu tibiae were endocortically treated with 1 M potassium hydroxide (KOH) solution for 1-14 days and then either mechanically tested in three-point bending, fatigued to failure or fatigued to induce stiffness loss. Computed Tomography scans, DXA, QUS, MRTA and three-point bend testing in the elastic region were performed on emu tibiae before and after either KOH treatment or fatigue to induce stiffness loss. Fracture surfaces were examined to determine failure mechanisms. Bone mineral and bone collagen were characterized using appropriate techniques. Bone mineral-collagen interface was investigated using Raman spectroscopy and atomic force microscopy (AFM). Endocortical KOH treatment does not affect bone mineral however, it causes in situ collagen degradation, rather than removal and may be weakening the mineral-collagen interface. These changes result in significantly compromised mechanical properties. Emu tibiae show significant decreases in failure stress and increased failure strain and toughness, with increasing KOH treatment time. The significant increase in toughness of KOH treated bones is due to structural alterations that enhance the ability of the microstructure to dissipate energy during the failure process, thereby slowing crack propagation, as shown by fracture surface analysis. KOH treated samples exhibit a lower fatigue resistance compared to untreated samples at high stresses only for both sexes. Partial fatigue testing results in similar decreases in modulus for all groups and sexes. The MRTA detected these changes whereas DXA and QUS did not. MRTA detects changes in bone mechanical properties induced by changes in collagen quality and fatigue and could be a more effective tool for predicting fracture risk.

Submitted by Andrea Maselli (deamaselli@yahoo.com.br) on 2018-01-19T20:23:34Z No. of bitstreams: 2 Tese PDF Impressão.pdf: 1825558 bytes, checksum: 52a8a536c671378887d7f638e8244a82 (MD5) Tese PDF Impressão.pdf: 1825558 bytes, checksum: 52a8a536c671378887d7f638e8244a82 (MD5) / Approved for entry into archive by Silvana Alvarez null (silvana@ict.unesp.br) on 2018-01-23T19:31:17Z (GMT) No. of bitstreams: 1 maselli_a_me_sjc.pdf: 1719458 bytes, checksum: 9bbc488c5915b3ba270ef7fd508729ab (MD5) / Made available in DSpace on 2018-01-23T19:31:17Z (GMT). No. of bitstreams: 1 maselli_a_me_sjc.pdf: 1719458 bytes, checksum: 9bbc488c5915b3ba270ef7fd508729ab (MD5) Previous issue date: 2017-12-17 / A mensuração da degradação de colágeno radicular é um importante parâmetro para avaliar a progressão de cáries na dentina ou a eficácia de métodos terapêuticos na prevenção de lesões não cariosas. O objetivo deste trabalho foi avaliar a degradação do colágeno da dentina radicular frente a métodos de prevenção de cárie, utilizando o teste da Hidroxiprolina e a microradiografia. Foram obtidos 40 blocos de dentina radicular com aproximadamente 1,5 mm de profundidade x 6 mm de diâmetro, a partir de incisivos bovinos, os quais foram submetidos ao processo de desmineralização artificial em tampão acetato (pH=5), a fim de induzir a formação de uma lesão cariosa. Em seguida as amostras foram submetidas aos tratamentos preventivos de cárie radicular: 1) Clorexidina 0,12% 1 min, 2) Flúor neutro 2% 1 min, 3) Nd: YAG Laser- 60 mJ 10 s e 4) Água Deionizada (controle) 1 min. Após os tratamentos as amostras foram expostas à degradação pela enzima colagenase (Tipo VII, Produto No. C0773, Sigma-Aldrich, St. Louis, MO, USA) por um período de 5 dias. Ao final do desafio da colagenase, a solução contendo a enzima foi coletada para ser submetida ao teste da hidroxiprolina, para a mensuração da quantidade de colágeno degradado por meio de colorimetria em espectrofotômetro. Em seguida as mesmas amostras foram submetidas à mais 2 dias de desmineralização. Em complemento ao ensaio da hidroxiprolina as amostras foram expostas à microradiografia transversal para visualização e medição da área degradada. Uma análise descritiva dos dados obtidos foi realizada de modo a determinar o teste estatístico a ser aplicado. A análise de variância (ANOVA) para a HYP revelou que não houve diferença estatisticamente significativa entre os tratamentos empregados (p>0,05). Os dados obtidos na microradiografia foram submetidos ao teste de Kruskal Wallis e teste de comparações múltiplas, Dunn. Os dados de reptetição, comparações entre a desmineralização inicial (5 dias) e a segunda desmineralização (2 dias), foi realizado individulamente em cada grupo por teste T de medidas repetidas ou Wilcoxon. Houve diferença significativa entre os grupos (p<0,0001). Dos tratamentos empregados, a clorexidina e o flúor foram eficazes na prevenção da progressão da cárie radicular. / Quantification of collagen degradation is an important parameter to evaluate dentin caries progression of caries prevention aid. The aim of this study was to evaluate root collagen degradation against preventive methods by using the hydroxyproline assay and microradiography technique. Forty root dentin blocks were obtained with 1,5x6 mm (depth x diameter) from bovine incisors, which were submitted to artificial demineralization process by acetate buffer (PH=5), in order to induce a carious lesion. Then, the samples were submitted to preventive therapeutic treatment of root caries: 1) Chlorhexidine 0,12% 1 min, 2) Fluoride 2% 1 min, 3) Nd:YAG Laser - 60 mJ 10s, 4) Deionized Water (control) 1 min. After that, the samples were exposed to degradation by the collagenase enzyme (Type VII, Product No. C0773, Sigma- Aldrich, St. Louis, MO, USA) for 5 days. Following the collagenase challenge, the enzyme solution was collected for assaying hydroxyproline released from collgen matrix, where it is possilble to measure the amount of degraded collagen by colorimetry in a spectrophotometer. Soon after, the same samples were submitted to a further 2 days of demineralization process. In addition to the hydroxyproline assay the samples were exposed to the transverse microradiography for a visualization of the degraded area. Soon after the colorimetric test the same samples were submitted to further two days of demineralization. A descriptive analysis of the data will be performed to determine the statistical test to be applied. ANOVA test revealed that there was no difference between the treatments (p>0,05). The microradiography data were submitted to the Kruskal Wallis test and multiple comparison test, Dunn. The repetition data, comparisons between the initial (5 days) and the second (2 days) demineralization, were performed individually in each group by repeated measures T-test or Wilcoxon (p<0,0001). Among the proposed treatments, chlorhexidine and fluoride were effective in preventing root caries progression.

Abstract Cancer progression is a multistep process dependent on tumour-stroma interactions. Various cell types, such as fibroblasts, endothelial, inflammatory and stem cells, as well as extracellular matrix (ECM) proteins, such as collagens, contribute to the tumour outcome. Tumour growth and invasion is accompanied by the proteolysis of ECM components mediated by various enzymes, such as matrix metalloproteases (MMPs). Proteolytic fragments released into the circulation may reflect cancer progression. The aim of this study was to develop novel in vitro methods for investigating cell invasion and for measuring cancer-associated collagen degradation. Human carcinoma cell invasion studies in vitro are often performed in organotypic cell culture models, mainly composed of rat or mouse ECM proteins. To create a human microenvironment for invasion studies, a novel organotypic model based on human uterine myoma (benign tumour) tissue was developed. Compared to the conventional collagen-based organotypic model, in the myoma model the carcinoma cell invasion depth was about eightfold and the invasion resembled, to a greater degree, the invasion pattern of dissected tissue samples of cancer patients. In addition, the invasion was easily quantified with a novel radioimmunoassay measuring type III collagen degradation products from the organotypic culture media. As human mesenchymal stem cells (MSCs) are one of the stromal cell types that may affect tumour progression, the mechanisms of stem cell invasion were also studied. On the surface of MSCs, Toll-like receptor 9 (TLR9) functions in immune defence against microbes. The activation of TLR9 with microbial DNA-resembling molecules induced human MSC invasion into the myoma tissue in a MMP-13-mediated fashion. To analyse cancer-associated soft tissue degradation, a novel enzyme immunoassay was developed. This novel assay enabled, for the first time, the measurement of type III collagen degradation products from human serum samples. In head and neck cancer patient sera, high levels of type III and type I collagen degradation products were shown to predict poor survival. In conclusion, the novel myoma model showed that the tumour microenvironment crucially affects carcinoma cell invasion. In addition, cancer-associated type III collagen degradation was successfully measured in cell cultures and in human sera by novel immunoassays. / Tiivistelmä Syövän eteneminen on monivaiheinen tapahtuma, jossa syöpäsolut ovat vuorovaikutuksessa lähiympäristönsä kanssa. Ympäristön eri solutyypit, kuten kantasolut ja sidekudoksen fibroblastit sekä soluväliaineen proteiinit kuten kollageenit, vaikuttavat syöpäsolujen invaasioon eli tunkeutumiseen ympäröivään kudokseen. Syövän invaasiossa useat entsyymit, mm. matriksin metalloproteaasit (MMP:t), hajottavat soluväliainetta. Kasvaimen kehittymisen aikana verenkiertoon vapautuu soluväliaineen hajoamistuotteita, joiden määrä voi kuvastaa sairauden etenemistä. Väitöstutkimuksen tarkoituksena oli kehittää uusia menetelmiä solujen invaasion ja syöpään liittyvän kollageenin hajoamisen tutkimiseen. Ihmisen karsinoomasolujen invaasion tutkimuksessa on perinteisesti käytetty kolmiulotteisia soluviljelymalleja, jotka koostuvat pääasiassa rotan tai hiiren soluväliaineproteiineista. Työssä kehitettiin uusi viljelymalli, jossa soluja kasvatettiin ihmisen hyvälaatuisen kohtukasvainkudospalan eli myooman päällä. Perinteiseen kollageenimalliin verrattuna myoomamallissa karsinoomasolut tunkeutuivat noin kahdeksan kertaa syvemmälle, ja solujen kasvu muistutti enemmän potilaiden syöpäkudosnäytteissä havaittua kasvutapaa. Invaasion voimakkuuden määrittämiseen kehitettiin vasta-aineisiin perustuva menetelmä, jolla mitattiin soluviljelmän kasvatusliuokseen myoomakudoksesta vapautuneiden tyypin III kollageenin hajoamistuotteiden määrää. Koska kantasolujen tiedetään voivan vaikuttaa syöpäkasvaimen leviämiseen, tutkimme myös ihmisen luuytimen kantasolujen invaasiota. Kantasolujen pinnalla TLR9-reseptori osallistuu immuunipuolustukseen mikrobeja vastaan. Kun reseptoria aktivoitiin mikrobi-DNA:ta muistuttavilla molekyyleillä, kantasolut alkoivat invasoitua myoomakudokseen ja MMP-13:n aktiivisuus soluissa lisääntyi. Syöpään liittyvän pehmytkudoksen hajoamisen tutkimiseksi kehitettiin vasta-ainemenetelmä, jolla onnistuttiin ensi kertaa mittaamaan potilaiden seeruminäytteistä tyypin III kollageenin hajoamistuotteita. Pään ja kaulan alueen syöpäpotilailla korkean tyypin III kollageenin hajoamistuotepitoisuuden todettiin liittyvän huonoon ennusteeseen. Tutkimus osoitti, että kasvaimen ympäristö vaikuttaa olennaisesti syöpäsolujen leviämiseen. Syöpään liittyvää tyypin III kollageenin hajoamista pystyttiin työssä kehitetyillä menetelmillä mittaamaan sekä soluviljelmistä että potilaiden seeruminäytteistä.