A Pseudoartrose Congênita da Tíbia (PCT) é uma das doenças mais desafiantes da ortopedia pediátrica pela dificuldade em obter a união óssea e, quando esta ocorre, em mantê-la. É uma doença muito rara, difícil de tratar devido à sua falta de conhecimento sobre a patogênese. As Células estromais mesenquimais multipotentes (CMM) podem desempenhar um papel na patogênese do PCT, possivelmente devido à falha da diferenciação osteogênica. O estudo das CMM pode ajudar a compreender a patogênese da doença e desenvolver novas estratégias terapêuticas baseados no uso desta célula no futuro próximo. Frente ao exposto, este trabalho teve como objetivo a análise das características biológicas das CMM isoladas de diferentes regiões anatômicas de medula óssea de pacientes com PCT. Para isto, amostras de medula óssea foram coletas a partir de locais afetados e não afetadas pela doença: Crista ilíaca do membro não afetada (CINA), crista ilíaca do membro afetada (CIA), tíbia não afetada (TNA), e tíbia afetada (TA). O numero de pacientes incluídos no estudo foi três: PCT1, PCT2 e PCT3. Os resultados mostraram que todas as células isoladas de pacientes com PCT apresentavam características compatíveis com as CMM. A taxa de formação de unidades formadoras de colônias das células da TA tanto no PCT2 quanto no PCT3 foi significativamente menor em relação às células da TNA e CINA respectivamente (p<0.05). A quantidade de células positivas para o marcador CD146 foi menor nas células da TA do PCT1 e PCT2, A análise estatística mostrou que não há uma diferença significativa. Este marcador esta relacionado com a capacidade multipotente e formação óssea in vivo. No PCT1 observou-se que formação de matriz mineralizada das CMM isoladas da CIA foi significativamente maior em relação a TA. Além disso, as células da TA do PCT1 observou-se um uma secreção significativa de alguns citocinas envolvidas no processo de formação óssea, como CCL2, CCL3, CCL4, TNA-alfa, PDGF-BB, e GM-CSF. A alteração destas citocinas pode levar a situações complicadas como o caso de não consolidação óssea. Com os resultados obtidos, se há demonstrado que as CMM da tíbia afetada tenta formar osso, mas no local da lesão é insuficiente, por tal motivo é preciso realizar estudos focados no mecanismo molecular. / Congenital pseudoarthrosis of the tibia (CPT) is one of the most challenging orthopedic diseases because of the difficulty in obtaining bone union and, when it happens, in maintaining it. It is a rare disease, difficult-to-treat due to the lack of knowledge about to pathogenesis. Multipotent mesenchymal stromal cells (MSC) may play a role in the pathogenesis of PCT, possibly due to a failure in the osteogenic differentiation. Studying these cells can help to better understand the pathogenesis of the disease and develop new therapeutic strategies based on the use of MSC in the near future. In view of the above, this work had the objective of analyzing the biological characteristics of CMM isolated from different anatomic regions of bone marrow of patients with PCT. For this, bone marrow samples were collected from sites affected and unaffected by the disease: unaffected limb iliac crest (CINA), affected limb iliac crest (CIA), unaffected tibia (TNA), and affected tibia (TA). The number of patients included in the study was three: PCT1, PCT2 and PCT3. The results showed that all cells isolated from PCT patients had characteristics compatible with CMM. The rate of formation of colonyforming units of TA cells in both PCT2 and PCT3 was significantly lower in TNA and CINA cells respectively (p <0.05). The amount of cells positive for the CD146 marker was lower in the TA cells of PCT1 and PCT2. Statistical analysis showed no significant difference. This marker is related to the multipotent capacity and bone formation in vivo. In PCT1 it was observed that the formation of mineralized matrix of CMCs isolated from CIA was significantly higher in relation to AT. In addition, PCT1 TA cells showed a significant secretion of some cytokines involved in the bone formation process, such as CCL2, CCL3, CCL4, TNA-alpha, PDGF-BB, and GM-CSF. The alteration of these cytokines can lead to complicated situations such as the case of non-consolidation of bone. With the results obtained, if the CMM of the affected tibia has been shown to try to form bone, but at the site of the lesion is insufficient, it is necessary to carry out studies focused on the molecular mechanism.

A Pseudoartrose Congênita da Tíbia (PCT) é uma das doenças mais desafiantes da ortopedia pediátrica pela dificuldade em obter a união óssea e, quando esta ocorre, em mantê-la. É uma doença muito rara, difícil de tratar devido à sua falta de conhecimento sobre a patogênese. As Células estromais mesenquimais multipotentes (CMM) podem desempenhar um papel na patogênese do PCT, possivelmente devido à falha da diferenciação osteogênica. O estudo das CMM pode ajudar a compreender a patogênese da doença e desenvolver novas estratégias terapêuticas baseados no uso desta célula no futuro próximo. Frente ao exposto, este trabalho teve como objetivo a análise das características biológicas das CMM isoladas de diferentes regiões anatômicas de medula óssea de pacientes com PCT. Para isto, amostras de medula óssea foram coletas a partir de locais afetados e não afetadas pela doença: Crista ilíaca do membro não afetada (CINA), crista ilíaca do membro afetada (CIA), tíbia não afetada (TNA), e tíbia afetada (TA). O numero de pacientes incluídos no estudo foi três: PCT1, PCT2 e PCT3. Os resultados mostraram que todas as células isoladas de pacientes com PCT apresentavam características compatíveis com as CMM. A taxa de formação de unidades formadoras de colônias das células da TA tanto no PCT2 quanto no PCT3 foi significativamente menor em relação às células da TNA e CINA respectivamente (p<0.05). A quantidade de células positivas para o marcador CD146 foi menor nas células da TA do PCT1 e PCT2, A análise estatística mostrou que não há uma diferença significativa. Este marcador esta relacionado com a capacidade multipotente e formação óssea in vivo. No PCT1 observou-se que formação de matriz mineralizada das CMM isoladas da CIA foi significativamente maior em relação a TA. Além disso, as células da TA do PCT1 observou-se um uma secreção significativa de alguns citocinas envolvidas no processo de formação óssea, como CCL2, CCL3, CCL4, TNA-alfa, PDGF-BB, e GM-CSF. A alteração destas citocinas pode levar a situações complicadas como o caso de não consolidação óssea. Com os resultados obtidos, se há demonstrado que as CMM da tíbia afetada tenta formar osso, mas no local da lesão é insuficiente, por tal motivo é preciso realizar estudos focados no mecanismo molecular. / Congenital pseudoarthrosis of the tibia (CPT) is one of the most challenging orthopedic diseases because of the difficulty in obtaining bone union and, when it happens, in maintaining it. It is a rare disease, difficult-to-treat due to the lack of knowledge about to pathogenesis. Multipotent mesenchymal stromal cells (MSC) may play a role in the pathogenesis of PCT, possibly due to a failure in the osteogenic differentiation. Studying these cells can help to better understand the pathogenesis of the disease and develop new therapeutic strategies based on the use of MSC in the near future. In view of the above, this work had the objective of analyzing the biological characteristics of CMM isolated from different anatomic regions of bone marrow of patients with PCT. For this, bone marrow samples were collected from sites affected and unaffected by the disease: unaffected limb iliac crest (CINA), affected limb iliac crest (CIA), unaffected tibia (TNA), and affected tibia (TA). The number of patients included in the study was three: PCT1, PCT2 and PCT3. The results showed that all cells isolated from PCT patients had characteristics compatible with CMM. The rate of formation of colonyforming units of TA cells in both PCT2 and PCT3 was significantly lower in TNA and CINA cells respectively (p <0.05). The amount of cells positive for the CD146 marker was lower in the TA cells of PCT1 and PCT2. Statistical analysis showed no significant difference. This marker is related to the multipotent capacity and bone formation in vivo. In PCT1 it was observed that the formation of mineralized matrix of CMCs isolated from CIA was significantly higher in relation to AT. In addition, PCT1 TA cells showed a significant secretion of some cytokines involved in the bone formation process, such as CCL2, CCL3, CCL4, TNA-alpha, PDGF-BB, and GM-CSF. The alteration of these cytokines can lead to complicated situations such as the case of non-consolidation of bone. With the results obtained, if the CMM of the affected tibia has been shown to try to form bone, but at the site of the lesion is insufficient, it is necessary to carry out studies focused on the molecular mechanism.

The local release of complexed siRNA from biomaterials opens precisely targeted therapeutic options. In this study, complexed siRNA was loaded to gelatin microparticles cross-linked (cGM) with an anhydride-containing oligomer (oPNMA). We aggregated these siRNA-loaded cGM with human mesenchymal stem cells (hMSC) to microtissues and stimulated them with osteogenic supplements. An efficient knockdown of chordin, a BMP-2 antagonist, caused a remarkably increased alkaline phosphatase (ALP) activity in the microtissues. cGM, as a component of microtissues, mineralized in a differentiation medium within 8–9 days, both in the presence and in the absence of cells. In order to investigate the effects of our pre-differentiated and chordin-silenced microtissues on bone homeostasis, we simulated in vivo conditions in an unstimulated co-culture system of hMSC and human peripheral blood mononuclear cells (hPBMC). We found enhanced ALP activity and osteoprotegerin (OPG) secretion in the model system compared to control microtissues. Our results suggest osteoanabolic effects of pre-differentiated and chordin-silenced microtissues.

Besides osteoconductivity and a high degradation rate, mesoporous bioactive glasses (MBGs) are specific for their highly ordered channel structure and high specific surface area, making them suitable as drug and/or growth factor delivery systems. On the other hand, the mesoporous channel structure and MBG composition can have an effect on common cell evaluation assays, leading to inconclusive results. This effect is especially important when MBG is mixed in composite bioinks, together with cells. Additionally, the hydrogel component of the ink can influence the degradation of MBG, leading to different ion releases, which can additionally affect the analyses. Hence, our aim here was to show how the MBG structure and composition influence common cell viability and differentiation assays when calcium (Ca)- or magnesium (Mg)-containing glass is part of an alginate-based composite bioink. We suggested pre-labeling of cells with DiI prior to bioprinting and staining with calcein-AM to allow identification of metabolically active cells expressing signals in both green and red channels, allowing the use of fluorescence imaging for cell viability evaluations in the presence of high amounts (7 wt %) of MBGs. The release and uptake of ions during degradation of CaMBG and MgMBG were significantly changed by alginate in the composite bioinks, as confirmed by higher release and uptake from bulk glasses. Additionally, we detected a burst release of Mg²⁺ from composites only after 24 h of incubation. Furthermore, we demonstrated that released ions and the mesoporous channel structure affect the measurement of lactate dehydrogenase (LDH) and alkaline phosphatase activity (ALP) in bioprinted composite scaffolds. Measured LDH activity was significantly decreased in the presence of CaMBG. On the other hand, the presence of MgMBG induced increased signal measured for the ALP. Taken together, our findings show how composite bioinks containing MBGs can interfere with common analyses, obtaining misleading results.

Eine bevorzugte Zellquelle für den Einsatz im Tissue Engineering sind mesenchymale Stammzellen (MSZ). Diese besitzen, neben einer hohen Proliferationsrate, die Fähigkeit, sich in verschiedene Zellen des mesodermen Ursprungs und in die entsprechenden Gewebetypen zu entwickeln. Um ein funktionales Gewebe zu erhalten ist es Ziel, sich bereits in vitro den in vivo Bedingungen anzunähern. Hierbei spielen neben der dreidimensionalen Struktur der Scaffolds auch die biochemische Mikroumgebung der Zellen in Form der unlöslichen extrazellulären Matrix (EZM) und den löslichen Mediatorproteinen wie Wachstums- und Differenzierungsfaktoren, sowie die physikalische Stimulation der Zellen eine wichtige Rolle. Während sich gegenwärtige Untersuchungen im TE vorwiegend mit den alleinigen Einflussfaktoren beschäftigen, verfolgt die vorliegende Arbeit das Ziel, die Auswirkungen kombinierter Stimuli durch Verwendung einer artifiziellen EZM, bestehend aus definierten Komponenten der nativen EZM, und physikalischer Stimuli durch elektrische Felder zu untersuchen. Letzteres erfolgte mit einem innerhalb der Arbeitsgruppe neu entwickelten System, dass die Stimulation von Zellen mit ausschließlich elektrischen Feldern, ohne störende Nebeneinflüsse, erlaubt.

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Eine bevorzugte Zellquelle für den Einsatz im Tissue Engineering sind mesenchymale Stammzellen (MSZ). Diese besitzen, neben einer hohen Proliferationsrate, die Fähigkeit, sich in verschiedene Zellen des mesodermen Ursprungs und in die entsprechenden Gewebetypen zu entwickeln. Um ein funktionales Gewebe zu erhalten ist es Ziel, sich bereits in vitro den in vivo Bedingungen anzunähern. Hierbei spielen neben der dreidimensionalen Struktur der Scaffolds auch die biochemische Mikroumgebung der Zellen in Form der unlöslichen extrazellulären Matrix (EZM) und den löslichen Mediatorproteinen wie Wachstums- und Differenzierungsfaktoren, sowie die physikalische Stimulation der Zellen eine wichtige Rolle. Während sich gegenwärtige Untersuchungen im TE vorwiegend mit den alleinigen Einflussfaktoren beschäftigen, verfolgt die vorliegende Arbeit das Ziel, die Auswirkungen kombinierter Stimuli durch Verwendung einer artifiziellen EZM, bestehend aus definierten Komponenten der nativen EZM, und physikalischer Stimuli durch elektrische Felder zu untersuchen. Letzteres erfolgte mit einem innerhalb der Arbeitsgruppe neu entwickelten System, dass die Stimulation von Zellen mit ausschließlich elektrischen Feldern, ohne störende Nebeneinflüsse, erlaubt.:1 Einleitung und Zielstellung 2 Theoretische Grundlagen 2.1 Der Knochen 2.1.1 Allgemeine Biologie und Physiologie des Knochengewebes 2.1.2 Knochenersatzmaterialien 2.2 Tissue Engineering von Knochengewebe 2.2.1 Trägermaterialien für das TE von Knochen 2.2.2 Zellen für das TE von Knochen 2.2.3 Artifizielle extrazelluläre Matrizes für das TE von Knochen 2.3 Einfluss elektrischer Felder auf Knochenumbauprozesse 2.3.1 Methoden zur Applikation von elektrischen Feldern 2.3.2 In vitro Untersuchungen zum Einfluss elektrischer Felder 2.3.3 Methode der Transformator-ähnlichen Einkopplung (TC) 3 Materialien 3.1 Technische Hilfsmittel und Geräte 3.2 Verbrauchsmaterialien 3.3 Chemikalien, Reagenzien und Kits 3.4 Antikörper 3.5 Oligonukleotide 3.6 Puffer-, Medien- und Lösungszusammensetzungen 3.7 Zellen 4 Methoden 4.1 Polycaprolacton-Co-Lactid (PCL)-Scaffolds 4.1.1 Präparation und Hydrophilisierung der PCL-Scaffolds 4.1.2 Beschichtung der PCL-Scaffolds 4.1.3 Charakterisierung der Beschichtung auf den PCL-Scaffolds 4.2 Zellkulturtechniken 4.2.1 Auftauen und Subkultivierung 4.2.2 Einfrieren 4.2.3 Induktion der osteogenen Differenzierung 4.2.4 Induktion der adipogenen Differenzierung 4.2.5 Induktion der chondrogenen Differenzierung 4.2.6 Besiedlung und Kultivierung der Zell-Matrix-Konstrukte 4.2.7 Elektrische Stimulation der Zell-Matrix-Konstrukte 4.2.8 Blockierung definierter Signaltransduktionswege 4.3 Mikroskopische Analytik der Zellen 4.3.1 Darstellung der Zellverteilung mittels Rasterelektronenmikroskopie (REM) 4.3.2 Qualitative Bestimmung von Fetttröpfchen mittels Oil-Red-O Färbung 4.3.3 Qualitative Bestimmung der Mineralisierung mittels vonKossa- Färbung 4.4 Durchflusszytometrie 4.5 Biochemische Analytik der Zellen 4.5.1 Bestimmung der Zellzahl mittels Lactatdehydrogenase (LDH)- Aktivität 4.5.2 Bestimmung der alkalische Phosphatase (ALP)-Aktivität 4.5.3 Quantitative Bestimmung des Kalziumgehaltes 4.6 Molekularbiologische Analytik / Genexpressionsanalyse 4.6.1 RNA Extraktion 4.6.2 cDNA-Synthese / Reverse Transkriptase PCR (RT-PCR) 4.6.3 Amplifikation von cDNA mittels quantitativer Real-Time PCR (qPCR) 4.7 Statistische Auswertung 5 Weiterentwicklung der Kammer zur TC-Einkopplung 5.1 Grundlegende theoretische Betrachtungen zur TC-Einkopplung 5.1.1 Ersatzschaltbild der TC-Einkopplung 5.1.2 Abschätzung des Eisenkernquerschnitts 5.1.3 Einfluss der Primärwindungszahl 5.2 Neudimensionierung und Aufbau der Stimulationseinrichtung 5.3 Verlauf der elektrischen Größen 5.3.1 Simulation 5.3.2 Messung 5.3.3 Abschätzung des magnetischen Feldes in der Kammer 5.4 Zusammenfassung 6 Zellexperimentelle Ergebnisse 6.1 Charakterisierung der humanen MSZ nach in vitro Kultivierung 6.1.1 Morphologie 6.1.2 Phänotypische Charakterisierung mittels Durchflusszytometrie 6.1.3 Multipotentes Differenzierungspotential 6.2 Zellverhalten auf den unbeschichteten PCL-Scaffolds 6.2.1 Ermittlung eines geeigneten Besiedlungsregimes 6.2.2 Zellverteilung und Proliferation der MSZ 6.2.3 Osteogene Differenzierung der MSZ 6.3 Einfluss der aEZM auf das Zellverhalten von MSZ 6.3.1 Quantitative Bestimmung der aEZM-Komponenten 6.3.2 Einfluss der aEZM auf die Adhärenz und Proliferation von MSZ 6.3.3 Einfluss der aEZM auf die osteogene Differenzierung von MSZ 6.4 Einfluss elektrischer Felder auf das Zellverhalten von MSZ 6.4.1 Einfluss der elektrischen Felder auf die Proliferation und osteogene Differenzierung von MSZ 6.4.2 Einfluss elektrischer Felder in Kombination mit Koll/sHya enthaltenden aEZM auf die Proliferation und osteogene Differenzierung von MSZ 6.4.3 Untersuchungen zu möglichen Signaltransduktionswegen 7 Diskussion der Ergebnisse 7.1 Charakterisierung der humanen MSZ nach in vitro Kultivierung 7.2 Zellverhalten auf den unbeschichteten PCL-Scaffolds 7.3 Einfluss der aEZM auf das Zellverhalten von MSZ 7.4 Einfluss elektrischer Felder auf das Zellverhalten von MSZ 8 Zusammenfassung und Ausblick Literaturverzeichnis Danksagung Eigene Publikationen und Mitautorschaften A Zusatzinformationen für die quantitative RT-PCR A.1 Versuchsdesign der Genexpressionsanalysen A.2 Qualitätskontrolle der isolierten RNA