Bone formation occurs through two ossification processes, intramembranous and endochondral. Intramembranous ossification is characterized by the direct differentiation of stem cells into osteoblasts, which then create bone. Endochondral ossification involves an intermediate step, as stem cells first differentiate into chondrocytes and produce a cartilage anlage. The chondrocytes mature into hypertrophic chondrocytes, which transform the cartilage anlage into bone. Bone tissue engineering has predominantly mimicked intramembranous ossification, creating osteoblast-based grafts through the direct differentiation of stem cells. Though successful in specific applications, greater adoption of osteoblast-based grafts has failed due to incomplete integration, limited regeneration, and poor mechanical maintenance. To overcome these obstacles, inspiration was drawn from native bone fracture repair, creating tissue engineered bone grafts replicating endochondral ossification. Hypertrophic chondrocytes, the key cell in endochondral ossification, were differentiated from mesenchymal stem cell sources by first generating chondrocytes and then instigating maturation to hypertrophic chondrocytes. Conditions influencing this differentiation were investigated, indicating the necessity of prolonged chondrogenic cultivation and elevated oxygen concentrations to ensure widespread hypertrophic maturation. Comparing the bone production performance of differentiated hypertrophic chondrocytes to differentiated osteoblasts revealed that hypertrophic chondrocytes deposit significantly greater volume of bone mineral at a higher density than osteoblasts, albeit in a more juvenile form. When implanted subcutaneously, the hypertrophic chondrocytes stimulated turnover of this juvenile template into compact-like bone, whereas osteoblasts proceeded with processes similar to bone remodeling, generating spongy-like bone. Implanting these tissue engineered constructs into an orthotopic, critical-sized femoral defect saw hypertrophic chondrocyte-based constructs integrate quickly with the femur and facilitate the creation of significantly more bone, resulting in a successful bridging of the defect. The success of hypertrophic chondrocyte-based grafts in overcoming the failures of tissue engineered bone grafts demonstrates the potential of endochondral ossification inspired bone strategies and prompts its further investigation towards clinical utilization.

Orientador: Álvaro Francisco Bosco / Banca: Luiz Alberto Milanezi / Banca: Sebastião Luiz Aguiar Greghi / Resumo: Nos últimos vinte anos, o estudo da regeneração das estruturas periodontais influenciou o desenvolvimento das terapias ósseas regenerativas. A regeneração óssea guiada cria um ambiente adequado para que ocorra a regeneração de tecido ósseo através do selamento da área do defeito com uma membrana. Quando os defeitos assumem grandes proporções, se faz necessário o uso do enxerto ósseo, podendo ser feito com o autógeno, alógeno, xenógeno ou materiais aloplásticos. O objetivo deste trabalho foi avaliar, histologicamente, em cavidades cirúrgicas realizadas em tíbias de ratos, o papel da matriz dérmica acelular como membrana na cicatrização óssea, o papel das partículas de vidro bioativo na cicatrização óssea, e o papel das partículas de vidro bioativo protegidas pela membrana de matriz dérmica acelular na cicatrização óssea. Os resultados revelaram que a matriz dérmica acelular apresentou biocompatibilidade e que foi capaz de isolar a cavidade cirúrgica, comprovando sua capacidade de ser empregada como membrana no processo de cicatrização sem interferir, significativamente, com a formação óssea; que o vidro bioativo mostrou-se biocompatível, sendo que as partículas estavam interpostas ao tecido ósseo neoformado, acarretando menor grau de formação óssea, e que o emprego da matriz dérmica acelular como proteção do vidro bioativo não assegurou um resultado superior ao do grupo em que o vidro bioativo foi utilizado sem a proteção de uma membrana. / Abstract: In the last twenty years, the regeneration of the periodontal structures has influenced the development of the new regenerative bone therapies. The guided bone regeneration promotes favorable conditions for the healing of the bone tissue through sealing the problem area with a membrane. When the problem area takes large proportions, a bone graft is needed; it can be autogenic, alogenic, xenogenic or alloplastic materials. The aim of this paper is to evaluate, histologycally, in the surgical cavities performed in tibial of rats, the role of the particles of bioactive glass protected by the membrane of the acellular dermal matrix in the bone healing. The results presented that the acellular dermal matrix showed biological compability and that it was able to isolate the surgical cavity proofing its ability of being used as membrane in the healing process without significantly interfering with the bone regeneration; that the bioactive glass is biological compatible, and the particles were interposed in the neoformed bone tissue leading to a decreased in bone formation, and that the use of acellular dermal matrix as a protection of bioactive glass did not show better results when compared to the group in which the bioactive glass was used without the membrane protection. / Mestre

Orientador: Rosemary Adriana Chiérici Marcantonio / Resumo: O objetivo deste estudo foi avaliar o efeito do uso de diferentes substitutos sobre o reparo ósseo de defeitos críticos de calvárias de ratos com ou sem osteoporose. Para tanto, foram divididos em três estudos. O estudo 1 avaliou o efeito de diferentes biomateriais sobre o reparo de defeitos críticos de calota (DCC) de 40 ratos saudáveis que foram aleatoriamente divididos em 5 grupos com 8 animais, de acordo com o tipo de biomaterial utilizado para preencher os DCC: Grupo COA (coágulo); Grupo AUT (osso autógeno); Grupo OBD (osso bovino desproteinizado); Grupo HA/ TCP (cerâmica bifásica composta de hidroxiapatita e β-fosfato tricálcio); Grupo TCP (β-fosfato tricálcio). Foram executadas análise microtomográfica para avaliação do comprimento linear remanescente (DLL) do DCC e o volume dos tecidos mineralizados (MT) dentro do defeito nos períodos de 3, 7, 15 e 30 dias após cirurgia. Adicionalmente, foi executado análise histométrica para avaliar a composição do tecido ósseo reparado (% Osso e % Biomaterial) no período de 30 dias. O grupo COA apresentou o menor DLL e MT dentro do DCC e maior % osso do que os outros grupos. O grupo OBD apresentou maior volume de tecidos mineralizados e maior % biomaterial do que o grupo os grupos AUT e TCP. Os grupos OBD e AUT apresentaram maior % osso que o grupo TCP. O estudo 2 avaliou o efeito do enxerto com OBD carregado de estrôncio (Sr) na cicatrização óssea em DCC em 42 ratos saudáveis. Foram feitos 2 defeitos/rato, e um destes aleatoriament... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Bone defects are a pervasive complication arising from many clinical conditions, both mechanical and pathological. Current treatments for large bony defects focus on applying bone grafts or synthetic materials to the defect area. Cell-based—and especially stem-cell—therapies have advanced greatly thanks to increasing attention focused on their ability to generate new tissues in situ with biomechanical properties approaching that of native tissue, but they suffer from their own shortcomings as well. Exosomes have been shown to play critical roles in cell-signaling and tissue regeneration and are therefore potentially ideal therapeutic vehicles for treating bone defects. Exosomes are small microvesicles counted amongst stem cells’ paracrine factors capable of delivering nucleic acid and enzymatic protein cargoes in a targeted 2 manner. Our previous studies have shown that hMSC-Exosomes are both proliferative and chemotactic, inhibit inflammatory cytokine production, and suppress osteoclast differentiation. Our long term goal is to develop hMSC-Exosome as a clinical therapy for bone regeneration. The objectives of this study were to determine the ability of hMSC-Exosome to enhance bone healing in a rat calvarial defect model, and to further investigate the integrity of the exosome under certain storage conditions. The specific aims of this study were: 1) To determine the osteogenic potential of hMSC-Exosomes in rat calvarial defects, and 2) To determine the impact of variable storage conditions on the integrity of exosomes. To investigate in vivo regenerative potential, rats with surgically-created craniotomy defects were treated with hMSC-Exosome suspension via a collagen gel matrix. After 4 weeks, the calvaria were harvested and analyzed via micro-CT. Volumetric micro-CT analysis showed that hMSC-Exosome could significantly enhance center healing, structural integrity, and growth uniformity in a calvarial defect model. Western blot and TEM showed thorough destruction of surface protein markers and decreased membrane integrity in lyophilized exosome fraction; moderate progressive surface protein marker loss and aggregation were observed with increasing freeze-thaw cycles. In summary, hMSC-Exosome is a promising therapeutic for treatment of bone defects.

Orientador: Daniela Ponzoni / Banca: Mariza Akemi Matsumoto / Banca: André Luís da Silva Fabris / Resumo: Objetivo: Avaliar por meio da análise histomorfométrica e histológica o potencial osteocondutor dos biomateriais Bio-Oss® (Geistilich, Suécia), GenOx Inorgânico® (Baumer S.A, São Paulo, Brasil) e GenOx Inorgânico 700 (Baumer S.A, São Paulo, Brasil) no reparo ósseo de defeitos críticos em calvária de ratos. Métodos: Foram utilizados 48 ratos divididos em quatro grupos de acordo com o material de preenchimento. Grupo controle (GC) com o defeito preenchido somente com coágulo sanguíneo (n=12), GBO preenchido com Bio-Oss® (n=12), G300 preenchido com GenOx Inorgânico® (n=12) e o G700 com GenOx Inorgânico 700 (n=12). Na calvária de cada animal foi utilizada uma broca trefina de 5 mm de diâmetro interno para confecção de defeito central com 6 mm de diâmetro e a após a inserção dos biomateriais correspondentes. Sobre todos os defeitos foi sobreposta a membrana bovina Gen Derm®. Posteriormente, submetidos à eutanásia aos 30 dias e 60 dias pós-cirúrgicos. Após 30 e 60 dias, tais defeitos foram encaminhados para análise histológica e análise histométrica para avaliação das seguintes variáveis: neoformação óssea, presença de biomaterial, leucócitos mononucleares e polimorfonucleares e outros tecidos encontrados (de granulação e medular). Resultados: O grupo mais representativo para neoformação óssea foi GC. Aos 30 dias apresentou maior média de tecido ósseo maduro (75,8). Aos 60 dias não houve diferença estatística entre os grupos GC (64,9), GBO (32,9), G300 (45,3) e G700 (26,6). O GBO a... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Abstract :Objective:The aim of this study was to evaluate the osteoconductive potential of Bio-Oss® (Geistilich, Sweden), GenOx Inorganic® (Baumer SA, São Paulo, Brazil) and GenOx Inorganic 700 (Baumer SA, São Paulo, Brazil) in the bone repair of critical defects in rat calvaria. Methods: 48 rats were divided into four groups, submitted to euthanasia at 30 days and 60 postoperative days: the control group (CG) with the defect filled only with blood clot (n = 12), GBO filled with Bio-Oss® (n = 12), G300 filled with GenOx Inorganic® (n = 12) and G700 with GenOx Inorganic 700 (n = 12). In the calvaria of each animal a 5mm trephine drill was used to make a central defect with 6 mm diameter and after the insertion of the corresponding biomaterials. Gen Derm® membrane was superimposed over all defects. Subsequently, these cavities were sent to histological and histometric analysis to evaluate the following variables: bone neoformation, presence of biomaterial, mononuclear and polymorphonuclear leukocytes and other tissues found (granulation and medullary). Results:The most representative group for bone neoformation was GC. At 30 days presented a higher mean of mature bone tissue (75.8). At 60 days there was no statistical difference between the GC (64.9), GBO (32.9), G300 (45.3) and G700 (26.6) groups. GBO had a higher amount of biomaterial after 30 days (115.9) and 60 days (118.5). Conclusion:All bovine biomaterials tested showed to be biocompatible and osteoconductive,Among the b... (Complete abstract click electronic access below) / Mestre

Orientador: Leonardo Perez Faverani / Coorientador Roberta Okamoto / Banca: Letícia Helena Theodoro / Banca: Ana Paula Farnezi Bassi / Banca: Claudio Maldonado Pastori / Banca: Cassio Edvard Sverzut / Resumo: Este trabalho teve como objetivo avaliar o efeito da biomodulação e bioatividade no reparo ósseo de ratas ovariectomizadas submetidas à simulação de fraturas femurais. Sessenta e quatro ratas Wistar (Rattus novergicus), fêmeas, com 6 meses de idade, em que metade dos animais foram submetidos à ovariectomia bilateral (OVX) e a outra metade à cirurgia fictícia de ovariectomia (SHAM), e um período de 3 meses foi acompanhado para a verificar se a osteoporose estava presente. As ratas foram submetidas à simulação de fratura em ambos os fêmures e a fratura foi fixada com miniplaca e parafusos do sistema 1,5 mm. A metade das amostras tiveram miniplacas com texturização de superfície tratadas por oxidação com plasma eletrolítico (PEO), que ficou em contato com o "gap" reparacional e, a outra metade com miniplacas convencionais, com superfície (CONV). Estes grupos experimentais não foram submetidos à biomodulação (CO). Para o mesmo desenho experimental dos grupos, no ato operatório, após a fixação das fraturas, os demais grupos experimentais foram submetidos à fotobiomodulação por meio da irradiação do laser de baixa intensidade (LASER), durante 5 minutos. Aos 14 e 42 dias de pós-operatório, foi administrada calceína e alizarina, respectivamente, pela via intramuscular, na dose de 20 mg/kg de peso. A eutanásia de todos os animais foi realizada aos 60 dias de pós-operatório. Os grupos experimentais (SHAM/LASER/CONV; SHAM/CO/CONV; SHAM/LASER/PEO; SHAM/CO/PEO; OVX/LASER/CONV; OVX/CO/CONV... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to evaluate the effect of biomodulation and bioactivity on the bone repair of ovariectomized rats submitted to simulation of femoral fractures. 64 female adults Wistar rats (Rattus novergicus), which half of the animals was submitted to bilateral ovariectomy (OVX) and the other half to the dummy ovariectomy surgery (SHAM), and after 3-month the osteoporosis was induced. The rats were submitted to fracture simulation in both of the femurs and the fracture was fixed with miniplate and screws of the system 1.5 mm. Half of the samples had surface-textured miniplates treated by with plasma electrolytic oxidation (PEO), which was in contact with the repair gap, and the other half with conventional miniplates with surface (CONV). These experimental groups were not submitted to biomodulation (CO). For the same experimental design of the groups, after fracture fixation, the other experimental groups were submitted to photobiomodulation through low level laser (laser), for 5 minutes. At 14 and 42 postoperative days, calcein and alizarin, respectively, were administered intramuscularly at a dose of 20 mg / kg body weight. The animals were euthanized at 60 postoperative days. The experimental groups (SHAM/LASER/CONV, SHAM/CO/CONV, SHAM/LASER/PEO, SHAM/CO/PEO, OVX/LASER/CONV, OVX/CO/CONV, OVX/LASER/PEO), after euthanasia, had the regions of interest, initially scanned in computerized microtomography to evaluate the bone volume parameters [BV / TV] and bone qualit... (Complete abstract click electronic access below) / Doutor

Background: Bone-grafting materials are required in orthopaedic surgery to treat bone defects. Bone formation assessment is required for the development of new strategies and approaches and for quality assurance and quality control of currently available materials. Approaches to the assessment of bone formation are yet to be systematically established, quantified and standardized. Aims: the overall aim of this study was to establish a set of comprehensive quantitative approaches for the assessment of bone formation and to evaluate the role of osteoblastic cells, growth factors, and scaffolds on this process. Materials & methods: both in vitro and in vivo parameters for osteoblast phenotype and bone formation were tested in osteosarcoma cell lines, Saos-2 and U2OS cells, mesenchymal cell line, C2C12 cells, primary adipose derived stromal cells (ADSCs), platelet rich plasma (PRP), and morselized bone grafts. The in vitro parameters used were measurement of alkaline phosphatase (ALP) activity, detection of bone nodules and biomineralization, and quantification of immunocytochemistry and conventional RT-PCR of osteoblast genotyping. In vivo parameters involved ectopic bone formation in nude mice and nude rats and a tibial defect model in nude rats. Histomorphometric and quantitative immunohistochemical analyses were also performed. Results: The in vitro characterization and ectopic bone formation capabiltity of Saos-2 and U2OS cells have been established. Saos-2 cell line, which presents many osteoblast genotype and phenotype, is a stable positive control for both in vitro and in vivo bone formation assessments. The measurement of ALP activity in both solid and liquid phases has been standardized. Both the genotype and phenotype of osteoblast lineage cells has been quantitatively assessed during the capability testing of ADSCs and PRP. Quantitative assessment of new bone formation and related protein markers in vivo has been successfully established through the testing of the biological properties of gamma irradiated morselized bone grafts. Conclusion: A comprehensive knowledge of the assessment of bone regeneration and formation in vitro and in vivo has been integrated and developed through years of study. A whole set of in vitro and in vivo approaches for the assessment of bone formation has been modified and standardized to best suit the different clinical applications. This thesis provides an outline of both in vitro and in vivo bone formation assessment and their clinical applications.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

The present study was carried out to evaluate the osteoconductive nature of Bio-OssRTM (natural deproteinized bone mineral) when used as an onlay bone substitute in a Guided Bone Regeneration model. The lateral surface of the mandible was exposed bilaterally, in 8 rabbits. On one side of the mandible, two titanium chambers were filled with Bio-OssRTM material and the chambers were then firmly secured to the mandible using screws. The pores in the titanium chambers were covered with a layer of Bio-GideRTM material. On the opposite side of the mandible, chambers without Bio-OssRTM were placed on the lateral side of the mandible as a control. After a healing period of 3 months, histologic sections were obtained from each chamber. It was observed that new bone was generated in both test and control chambers to various degrees. In the test group, the newly generated bone was 18.41% and in the control group it was 5.31%. This difference was statistically significant. Thus, in this experiment, Bio-OssRTM was proven to be osteoconductive.

Objective. The objective of this study is to test the efficacy of the biodegradable mesh as an alternative fixation device to the titanium mesh for support of onlay particulate, bone and cancellous, marrow (PBCM) grafts. Two parameters were evaluated: (1) Bone graft volume maintenance; and (2) Calcified tissue surface area and distribution. Methods. 12 New Zealand white rabbits were used and divided equally into two groups. In both groups, endochondral, cancellous bone graft was harvested from the anterior ilium. The bone graft was then compressed and placed as an onlay onto the lateral aspect of the mandible using two chambers for each animal. In the control group, the chambers were made of titanium, however in the test group the chambers were made of poly L-lactide, polyglycolide and trimethylene carbonate mesh (Inion GTR(TM) Finland), and thereafter the chambers were fixed to the mandible. The animals were then sacrificed after eight weeks postoperatively, and then Micro-CT imaging was performed for the entire sample in order to determine the total volume of calcified tissue present under each chamber. In addition, Histologic sections were obtained from each chamber, and thereafter stained using Toluidine blue and Von Kossa for the purpose of histomorphometric analysis in order to determine the calcified tissue surface area. Results. When comparing the data from both the titanium (Ti) and the biodegradable mesh (PLA) groups, Micro CT analysis showed no significant statistical difference (P-value = 0.546) with regard to the percentage of bone found under the chambers (Ti 15.0% and PLA 13.83%). Interestingly, neither did the histomorphometric analysis show any significant statistical deference (P-value = 0.8272) with regard to the percentage of calcified tissue surface area (Ti 16.86% and PLA 16.17%). Moreover, this calcified tissue was also found to be evenly distributed in both groups. Conclusion. Biodegradable mesh made of poly L-lactic and polyglycolic acid copolymers appears to be an appropriate alternative to the Ti mesh for support of PBCM bone grafts. However, further clinical trails should be conducted to confirm these findings.