Resumo: Objetivou-se avaliar, por meio de exames radiográficos e histológicos, a função da resina de poliuretana derivada do óleo de mamona (Ricinus communis), na forma de grânulos, no tratamento de defeito ósseo segmentar induzido no rádio de coelhos, tendo como padrão comparativo o enxerto ósseo esponjoso autólogo. Foram utilizados 20 coelhos da raça Norfolk, fêmeas, com idade entre 12 e 14 meses e peso entre 4 e 5kg. Uma falha óssea segmentar de 1,0 cm de comprimento foi induzida na diáfise de ambos os rádios pela excisão de fragmento osteoperiosteal. No rádio esquerdo o defeito foi tratado com a resina de poliuretana e no direito com auto-enxerto esponjoso colhido do úmero proximal esquerdo. Para realização dos exames histológicos foram eutanaziados cinco animais aos 15 dias, 30 dias, 60 dias e 120 dias de pós-operatório. A regeneração óssea foi maior no defeito tratado com auto-enxerto esponjoso em todos os períodos de observação, sendo que, aos 120 dias de pós-operatório, estes estavam totalmente reconstituídos. Pela avaliação histológica o polímero de mamona atuou como preenchedor de espaço, minimizando a produção de tecido fibroso no local, além de não apresentar sinais de reabsorção em nenhum dos momentos de avaliação. Foi possível concluir que o polímero de mamona na forma de grânulos é biocompatível e osteointegrável, porém não apresenta a mesma capacidade de regeneração óssea do auto-enxerto esponjoso. / Abstract: The aim of the present study was to compare, by radiographic and histological analyses, the castor oil plant polyurethane in granules presentation, applied to a segmental bone defect, created in both radial diaphyses, to the cancellous bone autograft, in order to evaluate an alternative to bone defect healing. Norfolk adult female rabbits, with approximately 13 months of age and a mean body weight of 4.5kg were used. Thus, a one-cm segmental defect was created in both radial diaphyses. The defect in the left radius was filled with the castor oil plant polyurethane, and the right one, filled with cancellous bone autograft, collected from the left proximal humerus. The rabbits were euthanased at 15, 30, 60 and 120 days post-surgery (5 animals/period), in order to proceed the histological analysis. New bone formation was increased and accelerated in the defect treated with cancellous bone autograft along all periods of observation. In the last moment (120 days), the defects were totally reconstituted and remodeled. The polyurethane acted as a space filler, minimizing the local production of fibrous tissue. No granules degradation or reabsorb were detected, as well as any inflammatory reaction. Thus, it was possible to conclude that the castor oil plant polyurethane, in granules presentation, was biocompatible and osteointegrated, but did not show the same bone regeneration capacity of the cancellous bone autograft. / Orientador: Paulo Iamaguti / Coorientador: Sheila Canavese Rahal / Mestre

Abstract Bone morphogenetic proteins (BMPs) constitute a large family of osteoinductive proteins. Different BMPs are widely used in animal experiments and increasingly in the field of bone surgery. However, the sterilization of BMPs and the choice of a suitable mode of delivery, which binds and slowly releases BMP molecules, are still under intensive investigation. The aims of this study were to evaluate the effects of ethylene oxide and gamma sterilizations and different delivery systems on the osteoinductivity of reindeer BMP extract by using heterotopic and orthopic animal models. Ethylene oxide gas (Steri-Vac 4XL, temperature 29 °C, exposure time 4 h, concentration 860 mg/l) and gamma (doses of 3.15 or 4.15 Mrad) sterilizations were used. The tested delivery systems for reindeer BMP were collagen (Lyostypt®), gelatine capsule (no.1) and composites containing collagen combined with tricalcium phosphate (TCP) or hydroxyapatite (HA) or biphasic tricalcium phosphate-hydroxyapatite (TCP/HA) or biocoral (NC) frames. The injectability of reindeer BMP was tested by using injections containing a saline or gelatine vehicle. Osteoinductivity was evaluated as ectopic bone formation in the thigh muscle pouches of mouse hind legs. The induced new bone was evaluated based on the incorporation of 45Ca or calcium yield, radiographs and histological examination three weeks after the operations. The effect of gamma sterilization on the bone healing capacity of reindeer BMP extract was evaluated in a rabbit radial bone defect model in comparison with non-sterilized reindeer BMP extract and recombinant BMP-2. Bone healing was evaluated after eight weeks based on radiographs, mechanical tests and peripheral computerized tomography (pQCT). All BMP implants induced new bone in vivo visible in radiographs, but no bone formation was seen in the control implants without reindeer BMP. Gamma sterilization did not decrease significantly the osteoinductivity of reindeer BMP extract, except when administered as an injection containing gelatine vehicle. Ethylene oxide sterilization decreased significantly the osteoinductivity of reindeer BMP extract and was significantly inferior compared to gamma sterilization. Reindeer BMP combined with collagen or composite containing collagen and TCP/HA frame induced new bone significantly better than reindeer BMP combined with composite containing collagen and TCP frame. BMP injections with gelatine or saline vehicles induced new bone effectively. Injections were easy to handle and well tolerated by the mice. Reindeer BMP extract administered with collagen carrier healed the bone defect of the rabbit radius significantly better than control implants without reindeer BMP or no treatment and its effect was comparable with rhBMP-2 treatment.

Die Entwicklung innovativer Therapiekonzepte für die Knochenregeneration erfordert validierte segmentale Knochendefekt-Tiermodelle. Dabei ist das Mausmodell für die präklinische Testung von zentraler Bedeutung, jedoch fehlen in der wissenschaftlichen Literatur bislang Angaben zu validierten, extern stabilisierenden kritischen segmentalen Knochendefektmodellen an der immundefizienten Maus. Das Ziel dieser Arbeit war daher die Entwicklung und in vivo Evaluierung eines zuverlässigen und einfach zu handhabenden Modells für extern stabilisierte kritische Knochendefekte an der immundefizienten Maus. Dreißig männliche nu/nu-Mäuse (40,7±2,8 g, 95±2,6 d) wurden mittels Isofluraninhalation narkotisiert und anschließend ein externer Fixateur (MouseExFix, RISystem, AO Research Institute Davos, Schweiz) am rechten Femur angebracht. Femorale Knochendefekte der Länge 1 mm (n=10), 2 mm (n=10) und 3 mm (n=10) wurden erzeugt. Der Wundverschluss erfolgte mit Einzelknopfnähten. Röntgenaufnahmen wurden unmittelbar postoperativ und im Folgenden alle zwei Wochen innerhalb des Beobachtungszeitraums von zwölf Wochen angefertigt und im Hinblick auf Knochenregeneration und –fusion ausgewertet. Weiterhin wurden histomorphologische, histomorphometrische, immunhistochemische und µCT-Analysen zur dreidimensionalen und zellulären Beurteilung der Knochenheilung angefertigt. Alle Tiere überlebten die Operation. Sechs Tiere starben innerhalb des Beobachtungszeitraums als Folge von starkem Blutverlust (n=1), Infektion (n=1), Pinlockerung, welche die Euthanasie erforderlich machte (n=2) und durch Komplikationen bei der Anästhesie (n=2). Die µCT-Analyse nach zwölf Wochen zeigte, dass 3/8 der 1 mm-Defekte, 5/8 der 2 mm-Defekte und 8/8 der 3 mm-Defekte eine Pseudarthrose aufwiesen. Das mittlere Defektvolumen stieg signifikant (p<0,001) mit der Größe des Defektes und betrug 0,36±0,42 mm³ (1 mm-Gruppe), 1,4±0,88 mm³ (2 mm-Gruppe), bzw. 2,88±0,28 mm³ (3 mm-Gruppe). Die mittlere Defektgröße verringerte sich entsprechend um 77,6% (1 mm-Gruppe), 56,8% (2 mm-Gruppe), bzw. 28,6% (3 mm-Gruppe). Die histomorphologischen, histomorphometrischen und immunhistochemischen Analysen zeigten keine statistisch signifikanten Unterschiede zwischen den drei experimentellen Gruppen. Das verwendete MouseExFix-System ist ein zuverlässiges und einfach zu handhabendes Verfahren zur Stabilisierung eines kritischen segmentalen Knochendefekts an der immundefizienten Maus, wenn ein 3 mm-Defekt erzeugt wird. Das im Rahmen der Studie entwickelte und validierte murine extern stabilisierte, segmentale kritische Knochendefektmodell ermöglicht die präklinische Evaluierung von Konzepten zur lokalen Knochenregeneration inklusive der Verwendung allo- und xenogener Zellen.:1 Einleitung 1 1.1 Hintergrund 1 1.2 Das Mausmodell 2 1.3 Übersicht Tierversuche mit Knochendefekten 5 1.4 Frakturheilung 6 1.4.1 Allgemeines 6 1.4.2 Räumliche Gliederung 7 1.4.3 Expression von Proteinen der extrazellulären Matrix 8 1.4.4 Das Vier-Phasen-Modell der Frakturheilung 9 1.4.5 Das anabolisch/ katabolische Modell der Frakturheilung 12 1.4.6 Beeinflussung der Frakturheilung 12 1.4.7 Das Diamantkonzept 14 1.5 Osteosynthesesysteme 14 1.6 Pseudarthrosen 15 1.6.1 Definition 15 1.6.2 Ätiologie 16 1.6.3 Klassifikation 16 1.6.4 Therapie 17 2 Material und Methoden 19 2.1 Versuchstiere 19 2.2 Operationsvorbereitung 19 2.3 Operationsablauf 20 2.4 Postoperatives Vorgehen 27 2.5 Verlaufskontrolle 28 2.6 Entnahme der Präparate 29 2.7 Anfertigung der µCT-Aufnahmen 30 2.8 Anfertigung der histologischen Schnitte 30 2.8.1 Bearbeitung der Femora 30 2.8.2 verwendete Färbungen 31 2.9 Beurteilung der Schnitte 32 2.9.1 Histologische Beurteilung 32 2.9.2 Histomorphometrische Beurteilung 33 2.10 Statistik 33 3 Ergebnisse 34 3.1 Überlebensraten und Gewichtsverlauf 34 3.2 Röntgenauswertung 35 3.3 CT-Auswertung 38 3.4 Histologische Auswertung 41 3.5 Histomorphometrische Auswertung 44 3.5.1 TRAP 44 3.5.2 Osteocalcin 46 3.5.3 Osteopontin 47 3.5.4 Osteonectin 48 4 Diskussion 50 4.1 Diskussion etablierter Modelle für kritische Knochendefekte 50 4.1.1 Diskussion der Konzeption der Modelle 50 4.1.2 Diskussion der durchgeführten Anästhesieverfahren 54 4.1.3 Diskussion der Ergebnisse 55 4.1.4 Diskussion der Defektlängen 56 4.1.5 Diskussion der verschiedenen Osteosynthesesysteme 57 4.1.6 Diskussion der Ausfaller 59 4.2 Anwendung und Nutzen immundefizienter Tiermodelle 60 4.3 Vergleich des tibialen und des femoralen murinen Frakturmodells 63 4.4 Diskussion der Beurteilung der Knochenheilung mittels bildgebender und histologischer Verfahren 63 4.5 Anwendungsmöglichkeiten 65 4.6 Schlussfolgerungen 66 5 Zusammenfassung 67 5.1 In deutscher Sprache 67 5.2 In englischer Sprache 68 6 Literaturverzeichnis 70 7 Anhang 92 7.1 Danksagung 92 7.2 Lebenslauf 93 7.3 Veröffentlichungen 95 7.4 Wertetabellen 96 7.5 Erklärungen zur Eröffnung des Promotionsverfahrens 109 / The development of innovative therapies for bone regeneration requires the use of advanced site-specific bone defect small animal models. In this context, murine models are of major importance as they allow for sufficient sample sizes prior to preclinical testing using larger animals. Owing to the small dimensions of the murine femur only a few custom fabricated fixation devices have been described in the literature so far. The aim of this investigation was to develop and validate a new, externally fixated critical size bone defect model for immunodeficient mice. Thirty male nu/nu mice (40.7 ± 2.8 g, 95 ± 2.6 days old) were anesthetized by isoflurane inhalation and an external fixation device (MouseExFix, RISystem, AO Research Institute Davos, Switzerland) was attached to the right femur. Femoral bone defects of 1 mm (n=10), 2 mm (n=10) and 3 mm (n=10) were created. Wounds were closed without any additional treatment. X-ray films obtained immediately after surgery and every 2 weeks postoperatively during the 12 week postoperative observation period were evaluated for bony regeneration and fusion. Furthermore, histomorphology, histomorphometry, immunohistochemistry and µCT analysis were performed. All of the animals survived the operation. Twenty four out of 30 animals reached the twelfth postoperative week. µCT analyses after twelve weeks showed that 3/8 of the 1 mm defects, 5/8 of the 2 mm defects and 8/8 of the 3 mm defects remained as nonunions. The defect volume was 0.36 ± 0.42 mm³ (1 mm group), 1.40 ± 0.88 mm³ (2 mm group), and 2.88 ± 0.28 mm³ (3 mm group) (p<0.001, between all groups). The defect size decreased by 77.6% (1-mm group), 56.8% (2-mm group) and 28.6% (3-mm group) (p=0.152, between all groups). Our method using the MouseExFix device has proven to be a reliable and easy-to-handle external fixation system for the stabilization of critical-size segmental bone defects in immundeficient mice when 3 mm defects are generated. This mouse model allows for high-throughput translational evaluation of concepts for site-specific bone regeneration including strategies using allogenic and xenogenic cell types.:1 Einleitung 1 1.1 Hintergrund 1 1.2 Das Mausmodell 2 1.3 Übersicht Tierversuche mit Knochendefekten 5 1.4 Frakturheilung 6 1.4.1 Allgemeines 6 1.4.2 Räumliche Gliederung 7 1.4.3 Expression von Proteinen der extrazellulären Matrix 8 1.4.4 Das Vier-Phasen-Modell der Frakturheilung 9 1.4.5 Das anabolisch/ katabolische Modell der Frakturheilung 12 1.4.6 Beeinflussung der Frakturheilung 12 1.4.7 Das Diamantkonzept 14 1.5 Osteosynthesesysteme 14 1.6 Pseudarthrosen 15 1.6.1 Definition 15 1.6.2 Ätiologie 16 1.6.3 Klassifikation 16 1.6.4 Therapie 17 2 Material und Methoden 19 2.1 Versuchstiere 19 2.2 Operationsvorbereitung 19 2.3 Operationsablauf 20 2.4 Postoperatives Vorgehen 27 2.5 Verlaufskontrolle 28 2.6 Entnahme der Präparate 29 2.7 Anfertigung der µCT-Aufnahmen 30 2.8 Anfertigung der histologischen Schnitte 30 2.8.1 Bearbeitung der Femora 30 2.8.2 verwendete Färbungen 31 2.9 Beurteilung der Schnitte 32 2.9.1 Histologische Beurteilung 32 2.9.2 Histomorphometrische Beurteilung 33 2.10 Statistik 33 3 Ergebnisse 34 3.1 Überlebensraten und Gewichtsverlauf 34 3.2 Röntgenauswertung 35 3.3 CT-Auswertung 38 3.4 Histologische Auswertung 41 3.5 Histomorphometrische Auswertung 44 3.5.1 TRAP 44 3.5.2 Osteocalcin 46 3.5.3 Osteopontin 47 3.5.4 Osteonectin 48 4 Diskussion 50 4.1 Diskussion etablierter Modelle für kritische Knochendefekte 50 4.1.1 Diskussion der Konzeption der Modelle 50 4.1.2 Diskussion der durchgeführten Anästhesieverfahren 54 4.1.3 Diskussion der Ergebnisse 55 4.1.4 Diskussion der Defektlängen 56 4.1.5 Diskussion der verschiedenen Osteosynthesesysteme 57 4.1.6 Diskussion der Ausfaller 59 4.2 Anwendung und Nutzen immundefizienter Tiermodelle 60 4.3 Vergleich des tibialen und des femoralen murinen Frakturmodells 63 4.4 Diskussion der Beurteilung der Knochenheilung mittels bildgebender und histologischer Verfahren 63 4.5 Anwendungsmöglichkeiten 65 4.6 Schlussfolgerungen 66 5 Zusammenfassung 67 5.1 In deutscher Sprache 67 5.2 In englischer Sprache 68 6 Literaturverzeichnis 70 7 Anhang 92 7.1 Danksagung 92 7.2 Lebenslauf 93 7.3 Veröffentlichungen 95 7.4 Wertetabellen 96 7.5 Erklärungen zur Eröffnung des Promotionsverfahrens 109

A new model of a critically-sized segmental femoral bone defect in rats was developed to enable in vivo imaging and facilitate post-mortem mechanical testing of samples. The critically-sized nature of the model was assessed and confirmed. The efficacy of sustained co-delivery of osteogenic (BMP-2 and TGF- Ò3) and angiogenic (VEGF) growth factors in promoting functional bone repair was assessed. Effects of scaffold modification in terms of geometry and composition were evaluated. The results indicated that co-delivery of BMP-2 and TGF- Ò3 resulted in a dose-dependent improvement in functional bone repair. Modification of the polylactide scaffold to include an absorbable ceramic component and a cored out geometry enhanced rate of union. Addition of VEGF to the scaffold treatment did not significantly impact revascularization of the defect site or functional repair of the bone defect. These data demonstrate that the complex environment of an acute bone defect requires different treatment strategies than simple ectopic models would suggest. A positive predictive correlation between bone repair parameters measured in vivo and mechanical functionality was established. The novel defect model demonstrated robustness and reproducibility. Implications for further research are discussed.

In the first part of these studies, we have performed standard microsurgical procedures provide a solution for long standing bone and soft tissue defects, even in cases of longstanding osteomyelitis of long bones. When long bony segments are missing, the microvascular bone transfer provides a reliable method. In smaller soft tissue and bone defects, the application of a descending genicular osteomyocutaneous flap provides an option with low donor site morbidity. In the second part, we have focussed on reducing the donor site morbidity and expanded on the application of tissue engineering methods. MSCs derived from bone marrow can be injected percutaneous or be combined with an autologous bony scaffold for treatment of delayed union and nonunion. The outcome of our studies, however, limited in number of patients, clearly showed the possibilities and advantages of this new approach. A multimodality approach is essential, but it can provide promising solutions. Well-established microvascular and modern biotechnology methods will improve patient satisfaction and functional recovery in severe limb trauma, often the result of high-energy motorcycle accidents. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

A reparação de defeitos ósseos é uma preocupação constante em diversas áreas da odontologia. Vários materiais são usados com a finalidade de ajudar e acelerar esta reparação, tais como enxertos autógenos, xenógenos, membranas e alguns medicamentos, assim como a associação de alguns destes métodos. A sinvastatina, medicamento utilizado para redução de colesterol, em alguns estudos, tem demonstrado ação na estimulação de formação óssea. O objetivo do presente estudo foi avaliar a influência da associação da sinvastatina à matriz desmineralizada de osso bovino na reparação de defeitos ósseos em calvária de ratos. Foram confeccionados defeitos ósseos na calvária de 48 ratos, sendo um defeito 5mm de diâmetro em cada parietal do animal. Os ratos foram divididos em três grupos de acordo com o material utilizado: grupo controle, que não recebeu nenhum tipo de substância, grupo sinvastatina-matriz óssea desmineralizada, que recebeu uma associação de sinvastatina e matriz desmineralizada de osso bovino e grupo matriz óssea desmineralizada que recebeu somente matriz desmineralizada de osso bovino como material de enxertia. Os sacrifícios foram realizados após trinta e sessenta dias. Após o sacrifício as calvárias foram radiografadas em aparelho de raios-X digital para análise de densidade radiográfica em tons de cinza e foram submetidas à preparação histológica de rotina, para posterior análise histológica descritiva e histométrica da área de matriz óssea neoformada, utilizando-se programa computacional para análise de imagens. Os dados submetidos à analise estatística ANOVA a 5% demonstraram que os animais que receberam a associação de sinvastatina-MODB apresentaram, na análise histométrica a menor área de neoformação e na análise radiográfica a menor densidade óssea. Concluiu-se, de acordo com a metodologia utilizada, que a associação sinvastatina-MODB influenciou negativamente o reparo ósseo. / Bone defect healing is a constant concern to several areas of Dentistry. A great variety of materials is currently used to help in the healing process as well as to speed it up. Materials such as autografts, xenografts, membranes, some drugs, and the association of some of them are being used. Simvastatin, a substance used to reduce cholesterol levels, has shown, in some studies, a capacity to stimulate bone formation. The purpose of the present experimental study is to evaluate the influence of the association of sinmvastatin with bovine demineralized bone matrix on the healing of bone defects in rats calvariae. Bone defects were produced in the calvariae of 48 rats, thus each animal had a defect in each parietal bone measuring 5mm of diameter. The rats were grouped according to the graft material used: the control group which didnt receive any substance; the simvastatin-demineralized bone matrix group which received an association of simvastatin and bovine demineralized bone matrix; and the demineralized bone matrix group which received bovine demineralized bone matrix. The animals were sacrificed after thirty or sixty days. After the sacrifices, digital radiographies were taken of the calvariae in order to analyse the radiographic density in shades of gray. They were also submitted to routine histological preparation for future descriptive histological and histomorfometric analyses of the new formed bone matrix through the use of a software to analyse the images. The data submitted to statistical analysis ANOVA (5%) showed that the animals which received the association simvastatin-demineralized bone matrix presented the smallest density and area of new bone formation of the three groups. According to the methodology used, we concluded that the association simvastatin demineralized bone matrix influenced negatively the bone healing.

Bone formation and bone healing were studied in the mandible, tibia and skull bones in adult, healthy and estrogen deficient rabbits implanted with different bone substitutes. In the first study an evaluation of the differences in bone regeneration in and around solid (Alveograf *) and porous hydroxyapatite (Interpore 200*) was undertaken. The implant material was placed into experimentally made bone defects and in half of the defects hydroxyapatite was mixed with a fibrin sealant (Tisseel *). The material alone or mixed with Tisseel was also placed subperiostally in the mandible. The observation time was six month. No difference in bone regeneration was found between solid or porous hydroxyapatite granulas and the addition of Tisseel* did not seem to disturb the bone healing process. The implant material placed subperiostally did not induce bone formation nor did it provoke any bone resorption. The addition of Tisseel made the implant material much easier to handle and retain in the tissue during surgery. Bone healing around hydroxyapatite implants was also evaluated in the second study. Experimental cavities in the mandible and tibia were filled with hydroxyapatite in granules or blocks (Interpore 200*) but now with or without autolyzed, antigen-extracted, allogeneic bone (AAA). Also in this study Tisseel* was used to facilitate the handling of the material. All cavities implanted with AAA-bone, regardless of the combination with hydroxyapatite or Tisseel, demonstrated excessive bone formation resembling exostosis formation. Thus, hydroxyapatite, both as granules and blocks, can be successfully combined with AAA bone utilizing the bone inductive capacity of AAA bone. The same model was used to study the healing in ovariectomized animals in the third study. Bone cavities were implanted with or without AAA bone and left to heal. The results indicate that the osteoinductive capacity of AAA bone is in operation also in animals deprived of a normal estrogen production. The effect of using AAA bone prior to implant insertion was studied in paper four. The bone-implant contact was significant higher when AAA bone had been used. The implant stability did not seem to be affected. In paper five defects were made in skull and tibial bone in estrogen deficient animals. The deficiency of estrogen was confirmed through blood analysis, the decrease in the weight of uterus and bone mineral density. The whole body scanning with DEXA showed that the ovariectomized animals developed osteopenia. Various degree of bone formation was seen in the defects due to the influence of the bone inductive substance AAA bone. The studies indicate that a conductive material like hydroxyapatite in granules or blocks could be useful in oral reconstructive surgery. The combination with AAA bone enhanced the bone formation in calvarial and tibial bone in healthy and estrogen deficient animals. Tisseel* could be used to facilitate handling and retention of the material in the intended position during the healing process without negative effects.

Strategien zur Gewebsreparatur durch Zelltransplantate erfordern die Verfügbarkeit einer ausreichenden Menge von Zellen, die Schaffung konduktiver Mikrokulturbedingungen für die Integration und die Entwicklung des Implantats und die Entwicklung reproduzierbarer chirurgischer Technik für die klinische Anwendung des kultivierten Transplantats. In der vorliegenden Arbeit wurden mehrere Techniken der Zelltransplantation entwickelt und tierexperimentell erprobt. Unter Verwendung von Alginat wurde eine neue sequentielle Zellkulturtechnik für Knorpeltransplantate entwickelt. Der optimale Kompromiß zwischen der Matrixstabilisierung und einer ausreichenden Diffusionskapazität für die Zellfunktion wurde bei einer Mischung aus 0,6 % Alginat und 4,5 % Fibrin gefunden. Weitere untersuchte Matrixstrukturen zur Transplantation von Chondrozyten, wie die bioresorbierbaren Polymere, das Kollagen-Fibrin-Gel besitzen gegenüber der gegenwärtig kommerziell genutzten Methode hinsichtlich des chirurgischen Prozederes bei vergleichbaren histologischen Ergebnissen Vorteile. Die histomorphologischen Veränderungen und die Entwicklung des Transplantats in vivo werden durch die spezifischen Bedingungen der Transplantatumgebung beeinflußt. Dabei ist ein vollständiges zonales und sequentielles Remodeling von Knorpel-Knochendefekten nur bei nicht ausdifferenzierten Zellen (embryonale Chondrozyten, periostale Zellen) zu erkennen, da diese Zellen ein exzellentes chondrogenes und osteogenes Potential besitzen. Transplantate unter Verwendung von Chondrozyten zeigen dagegen nur eine sehr geringe Rekonstruktion des subchondralen Knochens. Periostale Zellen sind in vitro ohne Verlust des Phänotyps amplifizierbar und stellen daher eine optimale Zellquelle für das Tissue Engineering dar. Für das Bone Engineering ist die Kombination der osteokonduktiven Eigenschaften unterschiedlicher Trägermaterialien mit Zellen, die ein osteogenes Potential besitzen ein neuer Weg zur Optimierung des Prozesses der knöchernen Rekonstruktion, wie in Versuchen zur Rekonstruktion segementaler Ulnadefekte bei Kaninchen gezeigt werden konnte. Die Herstellung eines präossären stabilen aber formbaren Transplantats mit vielfältigen klinischen Einsatzmöglichkeiten ist unter Verwendung von biodegradierbaren Polymeren und von Fibrinbeads realisierbar. Der Einsatz von Wachstumsfaktoren, wie TGF-?1 und die zunehmenden Erkenntnisse zu den Zell-Zell- und Zell-Matrix-Interaktionen ermöglichen die verbesserte Generation ortsständigen Gewebes durch multipotente Zellen. Die immer komplexere und umfassendere Wiederholung der sich in der Ontogenese abspielenden Vorgänge durch die Techniken des Tissue Engineering, ermöglicht die Schaffung therapeutischer Optionen zur Behandlung von Knochen- und Knorpeldefekten, wo bisher keine existierten oder nur unzulänglich vorhanden waren. / Strategies for tissue repair by cell transplants require the availability of a sufficient amount of cells, the creation of conductive microculture conditions for the integration and development of the implant and the development of reproducible surgical techniques for the clinical application of the cultivated transplant. Within the frame of the present work, several techniques of cell transplantation were developed and tested by way of experiment. By using alginate, a new sequential cell culture technique was developed for cartilage transplants. The optimum compromise between the matrix stabilization and a sufficient diffusion capacity for the cell function was found with a mixture of 0.6 % of alginate and 4.5 % of fibrin. Further investigated matrix structures for the transplantation of chondrocytes, such as the bio-absorbable polymers, the collagen fibrin jelly show advantages compared with the method commercially applied at present regarding the surgical procedure with the gained histological results being comparable. The histomorphological changes and the development of the transplant within the living body are influenced by the specific conditions of the transplant environment. In this connection, a complete zonal and sequential remodeling of osteochondrodefects can only be detected for non-outdifferentiated cells (embryonic chondrocytes, periosteal cells) as these cells have an excellent chondrogenic and osteogenic potential. When using chondrocytes for transplants, however, the transplant only shows a very little restoration of the subchondral bone. Periosteal cells can be amplified in the living body without losing the phenotype, thus constituting an optimum cell source for tissue engineering. For the bone engineering, the combination of the osteoconductive properties of different carrier materials with cells having an osteogenic potential is a new way for optimizing the process of bone restoration as it was demonstrated in tests for the restoration of segmental ulnar defects occurring with rabbits. The generation of a preosteal stable, but mouldable transplant with manifold clinical possibilities of utilization can be realized by using biodegradable polymers and fibrin beads. The use of growth factors, such as TGF-?1, and the increasing knowledge of cell-cell and cell-matrix interactions enable the improved generation of stationary tissue by multipotent cells. The more and more complex and comprehensive repetition of processes going on in the ontogenesis by way of tissue engineering enables the creation of therapeutic options for the treatment of osteochondrodefects where hitherto none existed or just in a too small number.

<p>Bone formation and bone healing were studied in the mandible, tibia and skull bones in adult, healthy and estrogen deficient rabbits implanted with different bone substitutes. </p><p>In the first study an evaluation of the differences in bone regeneration in and around solid (Alveograf *) and porous hydroxyapatite (Interpore 200*) was undertaken. The implant material was placed into experimentally made bone defects and in half of the defects hydroxyapatite was mixed with a fibrin sealant (Tisseel *). The material alone or mixed with Tisseel was also placed subperiostally in the mandible. The observation time was six month. No difference in bone regeneration was found between solid or porous hydroxyapatite granulas and the addition of Tisseel* did not seem to disturb the bone healing process. The implant material placed subperiostally did not induce bone formation nor did it provoke any bone resorption. The addition of Tisseel made the implant material much easier to handle and retain in the tissue during surgery.</p><p>Bone healing around hydroxyapatite implants was also evaluated in the second study. Experimental cavities in the mandible and tibia were filled with hydroxyapatite in granules or blocks (Interpore 200*) but now with or without autolyzed, antigen-extracted, allogeneic bone (AAA). Also in this study Tisseel* was used to facilitate the handling of the material. All cavities implanted with AAA-bone, regardless of the combination with hydroxyapatite or Tisseel, demonstrated excessive bone formation resembling exostosis formation. Thus, hydroxyapatite, both as granules and blocks, can be successfully combined with AAA bone utilizing the bone inductive capacity of AAA bone.</p><p>The same model was used to study the healing in ovariectomized animals in the third study. Bone cavities were implanted with or without AAA bone and left to heal. The results indicate that the osteoinductive capacity of AAA bone is in operation also in animals deprived of a normal estrogen production.</p><p>The effect of using AAA bone prior to implant insertion was studied in paper four. The bone-implant contact was significant higher when AAA bone had been used. The implant stability did not seem to be affected.</p><p>In paper five defects were made in skull and tibial bone in estrogen deficient animals. The deficiency of estrogen was confirmed through blood analysis, the decrease in the weight of uterus and bone mineral density. The whole body scanning with DEXA showed that the ovariectomized animals developed osteopenia. Various degree of bone formation was seen in the defects due to the influence of the bone inductive substance AAA bone. </p><p>The studies indicate that a conductive material like hydroxyapatite in granules or blocks could be useful in oral reconstructive surgery. The combination with AAA bone enhanced the bone formation in calvarial and tibial bone in healthy and estrogen deficient animals. Tisseel* could be used to facilitate handling and retention of the material in the intended position during the healing process without negative effects. </p>

Made available in DSpace on 2016-08-17T18:39:36Z (GMT). No. of bitstreams: 1 3621.pdf: 12488772 bytes, checksum: 3b893af6e3b0ea869fd5eda12f270c8a (MD5) Previous issue date: 2011-02-24 / Financiadora de Estudos e Projetos / Several resources have been studied in order to accelerate the process of bone repair. Among these resources, bioactive materials and low level laser therapy (LLLT) have gained prominence. Several studies suggest that both resources are able of stimulating osteoblast proliferation and osteogenesis at the fracture site, promoting a greater deposition of bone mass, which is fundamental for the consolidation process. Within this context, this project aimed to assess the effects of LLLT (_ = 830nm), with the fluencies of 120J/cm ² and scaffold Biosilicate®, used associated or not, on consolidation of induced tibial bone defects in the rats. In this study it was used 40 male Wistar rats (3 months ± 250g) divided into four groups (with 10 animals each): group control bone defect without any treatment (GC), group bone defect irradiated with LLLT 830nm (GL); group bone defect treated with implantation of scaffolds Biosilicate ® (GB); group bone defect treated with implantation of scaffolds Biosilicate ® and LLLT 830nm (GBL). The animals were submitted to laser irradiation (Ga-As-Al, 830nm, 100mW) at a single point on the bone defect for eight sessions, on alternate days. The euthanasia of animals occurred at day 15 after surgery, 24 hours after the last laser treatment session. Morphological analysis revealed that the laser group, showed better tissue organization in relation to other groups. Furthermore, morphometric analysis revealed that the irradiated animals showed a higher amount of newly formed bone compared to the other groups. The expression of COX-2 and RUNX-2/CBFA-1 were higher in GB and GBL groups. Also, biomechanical analysis revealed no statistical differences among experimental groups. From the results obtained in this study, it is possible to suggest that both treatments had osteogenic potential 15 days after surgery, but the LLLT was more effective in bone repair when compared to the biomaterials, or even when the two treatment modalities were associated. / Vários recursos têm sido estudados com o intuito de acelerar o processo de reparação óssea. Dentre esses recursos, os materiais bioativos e a Terapia Laser de Baixa Intensidade (LLLT) vêm se destacando, vários estudos sugerem que ambos os recursos são capazes de estimular a proliferação de osteoblastos e a osteogênese no local da fratura, promovendo maior deposição de massa óssea, fundamental para o processo de consolidação. Dentro deste contexto, esse projeto teve como objetivo verificar os efeitos da LLLT (_ = 830nm), com fluência de 120J/cm² e do scaffold de Biosilicato®, utilizados independentemente ou associados na consolidação de defeitos ósseos induzidos em tíbias de ratos. Foram utilizados 40 ratos machos da linhagem Wistar (3 meses de idade ± 250 gramas) distribuídos em 4 grupos experimentais com 10 animais cada: grupo controle com defeito ósseo e sem tratamento (GC); grupo defeito ósseo tratado com Laser 830nm (GL); grupo defeito ósseo tratado com implante de scaffolds de Biosilicato® (GB); grupo defeito ósseo tratado com implante de scaffolds de Biosilicato® e Laser 830nm (GBL). Os animais foram submetidos a irradiação Laser (Ga-As-Al, 830nm, 100mW) em um único ponto sobre o defeito ósseo por oito sessões de tratamento, em dias alternados. A eutanásia dos animais aconteceu no 15º dia após a cirurgia, 24 horas após a última sessão de tratamento Laser. A análise morfológica revelou que o grupo Laser, apresentou melhor organização tecidual em relação os demais grupos experimentais. Além disso, a análise morfométrica evidenciou uma maior quantidade de osso neoformado no grupo tratado com Laser comparado aos animais dos demais grupos. A expressão à COX-2 e a RUNX-2/CBFA-1 mostrou-se mais intensa nos grupos GB e GBL e na análise biomecânica não houve diferença estatística entre os grupos experimentais. A partir dos resultados obtidos neste estudo, pode-se sugerir que ambos os tratamentos apresentaram potencial osteogênico 15 dias após a cirurgia, porém a Terapia Laser de Baixa Intensidade foi mais eficaz no processo de reparo ósseo, quando comparado ao biomaterial, ou mesmo quando as duas modalidades de tratamento foram associadas.