Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Insulin is a mitogen for many cells and commonly signals through the classical, mitogenic Raf- MEK-ERK or metabolic PB-kinase pathways. Insulin deficiency or type I diabetes causes severe osteopenia. Obese patients with type II diabetes or insulin resistance, a disease associated with defective insulin signaling pathways and high levels of circulating insulin, have increased or normal bone mineral density. The question of whether hyperinsul inemia preserves bone mass is frequently raised. However, there is still a lot of controversy on the role of insulin as an osteoanabolic agent and this question still remains unanswered. A critical role for insulin signaling in bone building osteoblasts has recently been demonstrated with IRS-l knock-out mice. These mice developed low-turnover osteopenia due to impaired proliferation and differentiation, stressing the importance of osteoblastic IRS-l for maintaining normal bone formation. In the present study it was found that insulin does function in vitro as an osteoblast mitogen. This was illustrated in three relatively immature osteoblast (MBA-15.4, -15.6 mouse and MG- 63 human) cell lines, which responded to insulin with significant increases in proliferation. In the MBA -15.4 preosteoblasts insulin stimulation of proliferation was comparable to the welldescribed mitogen, TPA. The UMR-I06 cell line expresses markers of differentiated osteoblasts, and was much less responsive to insulin treatment. The difference in proliferative potential may be due to differences between spontaneously transformed cell lines, or the stage of cell differentiation. UOI26, a MEKI/2 inhibitor and wortmannin, a PB-kinase inhibitor, were used to investigate the pathway used by insulin to signal and activate ERK and osteoblast proliferation. In MBA-15.4 mouse preosteoblasts, GF-containing FCS was completely dependent on MEK for DNA synthesis. In contrast, in both MBA-15.4 and more mature MBA-15.6 osteoblasts, insulininduced proliferation was resistant to the inhibitors alone or in combination. Higher MEKinhibitor concentrations had no effect, and proliferation was also increased by the inhibitors in several experiments. This indicated that the classical, insulin mitogenic pathway was not involved in MBA-15.4 proliferation. Wortmannin had no effect on either insulin- or 20% FCSstimulated proliferation, but inhibited activation of Akt/PKB, the metabolic downstream target of PI3-kinase. Insul in signal ing to ERK was both MEK-and PI3-kinase- dependent, but this had no effect on proliferation. In contrast, FCS-stimulated ERK activation and proliferation was almost completely dependent on MEK-ERK activation. Proliferative signaling in the MG-63 human osteoblastic cell line in response to insulin was partially dependent on MEK and partially dependent on PB-kinase. In contrast, signaling in response to the phorbol ester, TPA, was partially dependent on PI3K but totally dependent on MEK-ERK. This indicates that the signal converges on ERK, suggesting the involvement of a PB-kinase upstream of a dominant MEK-ERK pathway. The differences found here between mouse and human insulin mitogenic signaling pathways indicate that there may be species differences between osteoblast signaling pathways, with mouse cells being independent and human cells being dependent on MEK for DNA synthesis in response to insulin. The effects of glucocorticoids on insulin mitogenic signaling in osteoblasts were also investigated, because chronic long-term steroid use results in excessive bone loss. The PTP inhibitor, sodium orthovanadate, reversed GC-impaired TPA- and FCS- induced proliferation in MBA-1SA and MG-63 preosteoblasts. PTPs, such as SHP-l and PTP-IB, dephosphorylate and inactivate phosphorylated kinases. Both SHP-l and PTPlB associated with kinases in the mitogenic signaling cascade of MBA-lS.4 preosteoblasts growing rapidly in 10% FCS. Further, SHP-I co-irnmunoprecipitated with active, tyrosine phosphorylated ERK, which may indicate that it can dephosphorylate and inactivate ERK. However, since the MEK-ERK or PB-kinase pathways are not important in insulin-induced proliferation in mouse osteoblasts, the PTPs are unlikely to be role players in the negative regulation of this signaling pathway. This was confirmed by the finding that vanadate was unable to reverse GC-induced decreases in insulinstimulated DNA synthesis. This suggests that vanadate-sensitive PTPs may not be important in the negative regulation of insulin-induced mouse osteoblast proliferation, and provides further evidence of a novel insulin mitogenic pathway in the MBA-lSA but not MG-63 osteoblastic cell line. / AFRIKAANSE OPSOMMING: Insulien is 'n mitogeen vir baie selle en gelei na binding aan die insulien reseptor, intrasellulêre seine via die klassieke, mitogeniese Raf-MEK-ERK of die metaboliese PB-kinase seintransduksie pad. 'n Insulien gebrek of tipe I diabetes veroorsaak osteopenie. Vetsugtige pasiënte met insulien weestandigheid of tipe II diabetes, 'n siekte wat geassosieer word met foutiewe insulien seintransduksie en hoë vlakke van sirkuierende insulien, het verhoogde of normale been mineraal digtheid (BMD). Die vraag of hiper insulin ernie 'n verlies aan beenmassa teëwerk word dikwels gevra. Teenstrydigheid oor die rol van insulien as 'n osteo-anaboliese stof bestaan egter steeds en hierdie vraag bly dus onbeantwoord. Dat insulien seintransduksie wel 'n kritiese rol speel in beenvormende osteoblaste is onlangs bevestig in studies met muise waarvan die geen vir IRS-l uitgeslaan is. Hierdie muise ontwikkel 'n lae omset osteopenie weens verswakte proliferasie en differensiasie. fn hierdie studie is gevind dat insulien wel in vitro as 'n osteoblast mitogeen kan funksioneer. Dit is in drie relatief onvolwasse (MBA-15.4, -15.6 muis en MG-63 mens) sellyne geillistreer, deur betekenisvolle verhogings in insulien-geaktiveerde proliferasie. In MBA-15.4 preosteoblaste is die persentasie verhoging in insulien-gestimuleerde proliferasie vergelykbaar met dié van die bekende mitogeniese forbolester, TPA. Die UMR-I06 sellyn het kenmerke van gedifferensieerde osteoblaste, en was baie minder responsief op insulien behandeling. Die verskil in die proliferasie vermoë van die verskillende sellyne kan die gevolg wees van verskille wat bestaan tussen spontaan getransformeerde sellyne of die stadium van sel differensiasie. 'n MEK 1/2 inhibitor, UO126 en 'n PB-kinase inhibitor, wortmannin, is gebruik om die insulien seintransduksie pad noodsaaklik vir die aktivering van ERK en osteoblast proliferasie te bepaal. In MBA-1S.4 muis pre-osteoblaste, was fetale kalf SenlTI1(FKS)-geinduseerde DNA sintese totaal afhanklik van MEK. Beide die MBA-15.4 en die meer volwasse MBA-15.6 muis osteoblaste was weerstandig teen die inhibitors op hulle eie, of in kombinasie. Verhoogde MEK-inhibitor konsentrasies het geen verdere effek gehad nie en in verskeie eksperimente is 'n verhoging in preliferasie selfs waargeneem met MEK-inhibisie. Hierdie resultate dui aan dat die klassieke insulien mitogeniese pad nie betrokke is in MBA-I5.4 gestimuleerde selproliferasie nie. Wortmannin het geen effek gehad op insulien- of20% FKS-gestimuleerde DNA sintese nie, maar het wel die aktivering van PB-kinase se metaboliese teiken, AktJPKB geinhibeer. Insulien seintransduksie aktiveer dus ERK deur beide MEK en PB-kinase, maar het geen effek op proliferasie gehad nie. FKS-gestimuleerde ERK aktivering en proliferasie was totaal afhanlik van MEK-ERK aktivering. Insulien-geaktiveerde DNA sintese in die mens MG-63 osteoblaste was gedeeltelik afhanklik van beide MEK en PB-kinase. Alhoewel IPA ook PB-kinase kon aktiveer, was dit totaal afhanklik van MEK vir DNA sintese. Dit dui aan dat daar 'n PB-kinase stroom-op van 'n dominante MEK-ERK seintransduksie pad voorkom. Die verskille wat ons dus waargeneem het in insulien mitogeniese seintransduksie tussen muis en mens, kan aandui dat insuliengestimuleerde seintranduksie paaie kan verskil van spesie tot spesie. Dit is bevestig met die muisselle wat onafhanklik is en mens selle wat afhanklik is van MEK aktivering vir insuliengeaktiveerde DNA sintese. Kroniese, langtermyn steroied behandeling kan beenverlies veroorsaak en die effek van glukokortikoide (GK) op die insulien mitogeniese pad in osteoblaste is dus ook ondersoek. Natrium-ortovanadaat, 'n proteien tirosien fosfatase (PIP) inhibitor het GK-verlaagde proliferasie in repons tot beide IPA- en FKS behandeling herstel in MBA-lSA en MG-63 preosteoblaste. PIPs soos SHP-l en PIP-l B funksioneer deur gefosforileerde kinases te defosforileer en dus te inaktiveer. Beide SHP-l and PIP-lB kon assosieer met kinases in die mitogeniese insulien seintransduksie pad van vinnig groeiende MBA-IS A preosteoblaste in 10% FKS. Verder het SHP-I ook geko-immunopresipiteer met aktiewe, tirosien-gefosforileerde ERK, wat aandui dat SHP-I met ERK assosieer om dit te defosforileer en inaktiveer. Die MEKERK of PB-kinase paaie is nie belangrik vir insulien-geaktiveerde seintransduksie in muis osteoblaste nie. Dit is dus onwaarskynlik dat die PIPs 'n rol sal speel in die negatiewe regulering van hierdie seintransduksie paaie. Die ontdekking dat vanadaat nie glukokortikoiedverlaagde insulien-geaktiveerde DNA sintese kan herstel nie, toon dat vanadaat-sensitiewe PIPs nie 'n rol speel in insulien-geaktiveerde proliferasie in muisselle nie. Hierdie bevinding het verder bevestig dat 'n nuwe insulien mitogeniese pad in die MBA-ISA, maar nie die MG-63 selle moontlik bestaan.

Active targeting with controlled delivery of therapeutic agents to bone is an ideal approach for treatment of several bone diseases. Since bisphosphonates (BPs) are known to have high affinity to bone mineral and are being widely used in treatment of osteoporosis, they are well-suited for drug targeting to bone. For this purpose, bifunctional hydrazine-bisphosphonates (HBPs) with spacers of various lengths and lipophilicity were synthesized and studied. Crystal growth inhibition assays demonstrated that the HBPs with shorter spacers bound more strongly to bone mineral, hydroxyapatite (HA), than did alendronate. HBPs were also demonstrated to be non-toxic to MC3T3-E1 pre-osteoblasts. The targeted delivery of the HBP-conjugated model drug, 4-nitrobenzaldehyde, was demonstrated through hydrolysis of the hydrazone linkage at the low pH of bone resorption and wound healing sites. In another series of experiments, a method to orient proteins on HA surfaces was developed to improve protein bioactivity. Enhanced green fluorescent protein (EGFP) and β-lactamase were used as model proteins. These proteins have a Ser or Thr at their N-terminus, which was oxidized to obtain a single aldehyde group that was subsequently used for bonding HBPs of various length and lipophilicity through formation of a hydrazone bond. The amount of protein immobilized through various HBPs was determined and found not to be exclusively dependent on the length of HBPs. The enzymatic activity of HBP-immobilized β-lactamase, measured with cefazolin as substrate, was found to be higher than β-lactamase that was simply adsorbed on HA. In a third set of studies, HBPs were evaluated for delivering parathyroid hormone (PTH) to bone mineral to enhance cell responses for bone formation. PTH was oxidized and conjugated to HBPs, followed by targeting to bone wafers. In vitro bioassays demonstrated that HBP-targeted PTH stimulated greater synthesis of cAMP in pre-osteoblasts compared to surfaces with simply adsorbed PTH. HBPs were also found to have similar pro-apoptotic activity to widely used alendronate. Overall, HBPs can be used for drug delivery to bone and oriented immobilization of proteins and peptides, with or without anti-osteoclastic action, for a variety of applications including bone tissue engineering.

Defects in bone are a constant and serious problem. They occur as a result of high energy trauma, congenital conditions or are created surgically to treat bone tumours or infection. Currently the treatment for these conditions is awkward for the patient, takes a long time and has a high complication rate. An elegant solution would be to mend the bone defect using the patient own cells; osteoblasts or mesenchymal stem cells seeded onto a supportive material scaffold. For successful regeneration of bone structures, a scaffold production technique has to be adopted that can precisely control porosity, internal pore architecture and fibre thickness, as well as maximising media diffusion and optimising scaffold mechanical properties so that the scaffold can withstand bone bearing pressures. It would also be beneficial if the scaffold uniformly distributed surface strain along the fibres throughout the entire scaffold as this would encourage more even cell proliferation/differentiation in the structure. This was addressed by performing a series of finite element analyses on the computer aided design model where the mechanical properties of the natural or synthetic polymer used have been incorporated to yield an accurate strain profile of the entire scaffold. The process used here to generate the scaffolds is a Rapid Prototyping method that creates a three-dimensional object through the repetitive deposition of fibres in layers via extrusion. Due to the high accuracy and versatility of the extruder, the diameter of the pores can be precisely controlled to an accuracy of 10μm, in the manufactured scaffolds the pore size ranges from 100 to 300μm as that is what is found in trabecular bone. Natural and synthetic polymers were plotted which altered the biodegradability properties of the scaffold and the degrees of cell adhesion, proliferation and differentiation in the structure. Scaffolds were manufactured that demonstrated compatibility with cell adhesion, proliferation and osteogenic differentiation. On completion of the scaffolds, the latter were seeded with osteoblasts or marrow stromal cells and put into a mechanically stimulating bioreactor machine to induce a small strain in the scaffold; this was performed to encourage cell proliferation/differentiation. The structure was left until the osteoblasts or marrow stromal cells modified the scaffold through bone deposition. In-vivo experiments were then undertaken. Preliminary data indicated an effect of mechanical stimulation of the cell/scaffold construct on the degree of mineralization of cell matrix generated by human osteogenic cells.

Les affections ostéo-articulaires concernent des millions de personnes. L’ingénierietissulaire osseuse, associant cellules souches mésenchymateuses humaines (CSM) etmatériaux synthétiques, pourrait répondre aux besoins cliniques. Pour cela, les différentescomposantes de cette approche et leur association doivent être mieux étudiées pour la rendreutile cliniquement. Durant cette thèse, une première étude animale proche du cas cliniquenous a permis de définir les points à améliorer pour le traitement des pertes osseuses. Nousavons ainsi pu développer un milieu spécifique induisant une différenciation rapide etterminale des CSM en ostéoblastes. Par la suite, l’utilisation de particules de céramiquescomme support cellulaire nous a permis d’obtenir des hybrides riches en matriceextracellulaire. Cet environnement 3D biomimétique permet l’engagement spontané des CSMvers un phénotype ostéoblastique et l’obtention d’une quantité osseuse importante in vivo.L’ensemble de ces résultats met en évidence l’importance de l’environnement et du stade dedifférenciation cellulaire pour la formation osseuse par ingénierie tissulaire osseuse. / Osteo-articular disorders affect millions of people over the world. Bone tissueengineering, an approach combining human mesenchymal stem cells (MSC) and syntheticmaterials, could potentially fulfill clinical needs. However, the different components of thisapproach and their association should be investigated further to make it clinically useful. Inthis thesis, an initial animal study close to clinical situation allowed us to identify areas thatneed improvement for regenerating bone defect. We were then able to develop a specificmedium which induces a rapid and terminal osteoblastic differentiation of MSC.Subsequently, the use of ceramic particles as cell support has allowed us to obtain hybridmainly composed of extracellular matrix. This biomimetic 3D environment allowsspontaneous osteoblastic commitment of MSC and induces a large bone quantity in vivo.Overall, these results highlight the importance of the environment and the cell differentiationstate for bone formation using bone tissue engineering.

The demand for treatment strategies of musculoskeletal tissues is continuously growing, especially considering the increasing number of elderly people with degenerative diseases of the skeletal system. Despite major strides in the field of bone regenerative medicine during the years, current therapies, such as bone grafts, still have several limitations. Multipotent stem cells, such as mesenchymal stem cells (MSCs) are promising candidates for tissue repair because of their differentiation potential and their capacity to undergo extensive replication. However, isolating a homogeneous population of MSCs from multiple sources is an area that needs to be addressed. Also, the knowledge regarding the mechanisms and pathways that lead to the final osteogenic differentiation is still scarce. The following research is a feasibility study on a new isolation technique developed by our lab. The major focus of the research will be the isolation and characterization of mesenchymal stem cells from both adult bone marrow and umbilical cord blood using a novel isolation method based on immunodepletion. Furthermore we will look at the potential of these isolated MSCs to differentiate into mature, bone producing osteoblasts. The results of the studies showed that our novel isolation method allowed proliferation of a homogeneous MSC population. Our irnrnunodepleted MSCs were 99% double positive for antibodies CD44 and CD105 which are highly specific for multipotent MSCs while cells isolated using the plastic adherence method were only 43% double positive for the two MSC-specific markers. Homogeneous MSCs were derived from both adult bone marrow and umbilical cord blood using our isolation method. Utilizing the techniques of confocal microscopy, von Kossa staining, and RTPCR we also show that MSCs, upon stimulation with osteogenic supplements, differentiate into osteoblasts capable of being used for bone tissue engineering applications.

A não-união óssea devido a condições pós-traumáticas ou patologias apresentam grandes desafios para a prática ortopédica, onde a necessidade de analisar novos biomateriais que auxiliem no processo da regeneração, tem levado vários pesquisadores a estudarem a membrana do látex natural (LN) e colágeno (COL) como formas de tratamento através da regeneração óssea guiada. O uso do LN têm mostrado promissores resultados em aplicações biomédicas, apresentando características como biocompatibilidade, crescimento ósseo e estímulo natural à angiogênese. Estudos que utilizam as membranas de colágeno para a mesma finalidade também observaram bons resultados em relação à biocompatibilidade, função hemostática, poder de auxiliar na cicatrização de feridas, capacidade de estimular a adesão celular e quimiotaxia. Porém, possuem alto poder de se degradar antes de completar o período final de tratamento. Desta forma, a utilização de membranas contendo estes dois componentes pode auxiliar no processo de neoformação óssea através da regeneração óssea guiada (ROG), e o LN pode funcionar como matriz para a liberação controlada do colágeno no defeito. Portanto, o objetivo deste estudo foi avaliar o efeito da membrana mista, composta por LN/COL, no processo de reparação óssea em defeitos de tíbia de ratos. Para tanto, foram utilizados quarenta (40) ratos machos da raça Rattus norvegicus albinus, da variedade Wistar, com cinco semanas de idade e peso médio de 300 g. Foi realizado um defeito ósseo bilateralmente nas tíbias de todos os animais, que foram divididos em 4 grupos (n=10), sendo: grupo controle (C); e tratados: grupo látex natural (LN); grupo látex natural/colágeno (LN/COL) e grupo colágeno (COL). O tempo de tratamento foi de 42 dias e a eutanásia dos animais foi realizada por overdose de anestésico (ketamina/xilazina). Após a eutanásia, as tíbias de cada animal foram dissecadas e limpas das partes moles, e armazenadas de acordo com protocolo de cada análise. Foram realizadas análise de microtomografia computadorizada (micro CT), densitometria, ensaio mecânico, histologia, e reação de polimerização em cadeia quantitativo (RT-qPCR). A análise estatística dos dados foi realizada utilizando o programa SPSS® (Versão 20.0, IBM®, Armonk, EUA) e foi adotado nível de significância de 5% (p<= 0,05). Foi utilizado o teste de Shapiro-Wilk para testar se os dados apresentavam ou nãodistribuição normal. A comparação entre os grupos (dados paramétricos) foi realizada através do teste ANOVA com pós-teste de Tukey. Os dados não-paramétricos foram analisados através do teste de Kruskal-Wallis. Foram demonstradas diferenças estatísticas no BV entre os grupos LN e CON (p<0,008); BV/TV do grupo LN/COL comparado ao CON (p<0,016) e ao LN (p<0,016); na neoformação óssea entre CON e COL (p< 0,002); e na força máxima quando comparado o grupo COL com CON (p<0,050) e (p<0,001). Os resultados demonstraram que não houve diferença significativa entre os grupos na análise da DMO (p=0,669), colágeno (p=0,224), rigidez relativa (p=0,461), RANK L (p=0,121), OC (p=0,066) e OPG (0,287), porém, todas as membranas utilizadas demonstraram valores superiores ao grupo CON. Sendo assim podemos concluir que as membranas utilizadas como tratamento apresentaram eficácia na reparação óssea de tíbia de ratos, no entanto, as membranas de COL e LN/COL demonstraram resultados mais significativos no período analisado. / The non-union of bone due to post-traumatic conditions or pathologies presents major challenges for orthopedic practice, where the need to analyze new biomaterials that aid in the regeneration process, has led several researchers to study the natural latex (LN) membrane and collagen (COL) as forms of treatment through guided bone regeneration. The use of LN has shown promising results in biomedical applications, presenting characteristics such as biocompatibility, bone growth and natural stimulation to angiogenesis. Studies that use collagen membranes for the same purpose have also observed good results in relation to biocompatibility, hemostatic function, the power to aid wound healing, the ability to stimulate cell adhesion and chemotaxis. However, they have a high capacity to degrade before completing the final treatment period. Thus, the use of membranes containing these two components can aid in the bone neoformation process through guided bone regeneration (ROG), and the LN can function as a matrix for the controlled release of the collagen in the defect. Therefore, the objective of this study was to evaluate the effect of the mixed membrane, composed of LN / COL, in the process of bone repair in rat tibia defects. For this purpose, forty (40) male Rattus norvegicus albinus rats of the Wistar variety, with five weeks of age and average weight of 300 g were used. A bone defect was performed bilaterally in the tibiae of all the animals, which were divided into 4 groups (n = 10), being: control group (C); and treated: natural latex group (LN); natural latex / collagen group (LN / COL) and collagen group (COL). The treatment time was 42 days and euthanasia of the animals was performed by anesthetic overdose (ketamine / xylazine). After euthanasia, the tibia from each animal were dissected and cleaned from the soft tissues, and stored according to each analysis protocol. Computed microtomography (micro CT), densitometry, mechanical assay, histology, and quantitative polymerization (RT-qPCR) were performed. Statistical analysis of the data was performed using the SPSS® program (Version 20.0, IBM®, Armonk, USA) and a significance level of 5% was adopted (p<=0.05). The Shapiro-Wilk test was used to test whether or not the data presented normal distribution. The comparison between the groups (parametric data) was performed through the ANOVA test with Tukey post-test. Nonparametric data were analyzed using the Kruskal-Wallis test. Statistical differences in BVwere found between the LN and CON groups (p <0.008); BV / TV of the LN / COL group compared to the CON (p <0.016) and the LN (p <0.016); in the bone neoformation between CON and COL (p <0.002); and maximal strength when compared to the COL group with CON (p <0.050) and (p <0.001). The results showed that there was no significant difference between groups in the analysis of BMD (p = 0.669), collagen (p = 0.224), relative rigidity (p = 0.461), RANK L (p = and OPG (0.287), however, all the membranes used showed higher values than the CON group. Thus, it can be concluded that the membranes used as treatment showed efficacy in rat tibia repair, however, the COL and LN / COL membranes showed more significant results in the analyzed period.

Técnicas para reconstruções ósseas são descritas em artigos científicos e dentre as barreiras mecânicas utilizadas, a malha de titânio vem demonstrando possibilidades de tratamento para ganho ósseo. Estudos pré-clínicos são escassos na literatura relatando a melhor morfologia de malha de titânio a ser utilizada, além da necessidade de uso de membrana oclusiva adicional. Dessa forma, o objetivo do estudo é avaliar se há diferença na qualidade e volume ósseo formado ao utilizar malhas de titânio com diferentes diâmetros de poro, e avaliar a necessidade de utilização de uma membrana adicional, sobre a malha de titânio. Para este estudo foram utilizados 28 ratos adultos machos do tipo Wistar, com peso médio de 410,8 gramas. Os animais foram divididos aleatoriamente em quatro grupos experimentais principais: Grupo P300: uso de malhas de titânio Painel Grade 15 Neodent®, com espessura de 0,3 mm e perfuração medindo 3 mm entre os vértices (n = 7); Grupo P175: uso de malhas de titânio Painel Grade 20 Neodent®, com espessura de 0,3 mm e perfuração com 1,75 mm diâmetro (n = 7); Grupo P85: uso de malhas de titânio Bionnovation® Surgitime Titânio, com espessura de 0,04 mm e perfuração com 0,85 mm de diâmetro (n = 7); Grupo P15: uso de malhas de titânio Bionnovation® Surgitime Titânio de espessura de 0,04 mm e perfuração com 0,15 mm de diâmetro (n = 7). Em todos os grupos, cada fêmur foi subdividido em teste (fêmur em que foi utilizado Bio-Oss Collagen® e membrana de colágeno BioGide®) e controle (apenas Bio-Oss Collagen®). Após 24 horas do procedimento cirúrgico, o qual foi realizado com anestesia geral, os animais foram submetidos a análise de microtomografia computadorizada in vivo, também sob anestesia. Após 30 dias, foram novamente submetidos a microtomografia computadorizada in vivo e, em seguida, eutanasiados para processamento histológico. Após análise estatística, foi observado que não houveram diferenças estatísticas em relação aos parâmetros volumétricos, nas comparações intra e entre grupos. Em relação a densidade mineral óssea, nas comparações intra grupos, relacionando fêmur teste e controle, não foram observadas significâncias estatísticas. Nas comparações entre grupos, foram observadas maior densidade nos grupos com maior diâmetro de perfuração (p<0,05). Nas análises histológicas, foi possível observar neoformação óssea do tipo esponjosa, demonstrando o mesmo padrão em todos os grupos, com presença de osteócitos em lacuna, início de um processo de amadurecimento ósseo com formação de lamelas concêntricas e íntima relação do novo osso formado pelo enxerto e o fêmur. De acordo com os resultados pode-se concluir que, o diâmetro do poro da malha de diâmetro pode interferir na qualidade óssea, porém, irá depender do enxerto ósseo utilizado, e o uso adicional de membrana de colágeno, quando associada a enxerto ósseo, não determinou a formação de novo osso de qualidade superior / Techniques for bone reconstruction are described in scientific articles and, among mechanical barriers used, titanium mesh has been showing possibilities of treatment for bone gain. Preclinical studies are scarce in the literature reporting the best morphology of titanium mesh to be used, in addition to the need for additional occlusive membrane. Thus, the objective of this study is to evaluate if there is a difference in bone quality and volume formed when using titanium meshes with different pore diameters, and to evaluate the need to use an additional membrane on the titanium mesh. For this study, 28 male Wistar male rats with an average weight of 410.8 grams were used. The animals were randomly divided into four main experimental groups: Group P300: use of titanium meshes Grid Panel 15 Neodent®, with thickness of 0.3 mm and perforation measuring 3 mm between vertices (n = 7); Group P175: use of titanium meshes Grid Panel 20 Neodent®, with thickness of 0.3 mm and perforation with 1.75 mm diameter (n = 7); Group P85: use of titanium meshes Bionnovation® Surgitime Titanium, 0.04 mm thick and 0.85 mm diameter (n = 7); Group P15: use of titanium meshes Bionnovation® Surgitime Titanium thickness 0.04 mm and perforation with 0.15 mm diameter (n = 7). In all groups, each femur was subdivided into test (femur in which Bio-Oss Collagen® and BioGide® collagen membrane was used) and control (Bio-Oss Collagen® only). After 24 hours of the surgical procedure, which was performed under general anesthesia, the animals were submitted to in vivo microtomography, also under anesthesia. After 30 days, they were again submitted to computerized in vivo microtomography and then euthanized for histological processing. After statistical analysis, it was observed that there were no statistical differences in relation to the volumetric parameters, in intra and inter group comparisons. Regarding bone mineral density, in intragroup comparisons, relating femur test and control, no statistical significance was observed. In the comparisons between groups, higher densities were observed in the groups with greater drilling diameter (p <0.05). In the histological analyzes, it was possible to observe new bone formation of the spongy type, showing the same pattern in all groups, with presence of osteocytes in the gap, beginning of a bone ripening process with concentric lamella formation and an intimate relation of the new bone formed by the graft and the femur. According to the results, it can be concluded that the pore diameter of the diameter mesh may interfere with bone quality, however, it will depend on the bone graft used, and the additional use of collagen membrane, when associated with a bone graft, does not determined the formation of new bone of superior quality

Considerando as diferentes aplicações clínicas dos enxertos ósseos nas reconstruções alveolares e a dificuldade de se obter ganhos ósseos em altura, o presente estudo avaliou do ponto de vista histológico a integração do enxerto autógeno (AU), do aloenxerto ósseo liofilizado desmineralizado (ALD), do aloenxerto ósseo congelado mineralizado (ACM) e do coágulo sanguíneo (CO) em um modelo de regeneração óssea vertical. Foram utilizados nove coelhos, sendo um animal doador primário de enxertos ósseos e oito animais submetidos a um modelo de regeneração óssea guiada (ROG), onde 32 cilindros de titânio foram fixados na calota craniana e preenchidos aleatoriamente com AU, ALD, ACM e CO. Após 13 semanas, os animais sofreram eutanásia e o conteúdo dos cilindros submetido à avaliação histológica e histomorfometrica para quantificar a área total de tecido neoformado (AT), o osso neoformado (ON) e o remanescente do material enxertado (MR). Os dados foram submetidos aos testes t-Student e Mann-Whitney com nível de significância de 5%. Os resultados mostraram que em relação à AT os valores médios foram significantes para ACM e ALD e seguiram a seguinte relação: ACM = ALD > AU > CO. Para a variável neoformação óssea as intervenções ALD e ACM mostraram maior quantidade de tecido ósseo formado do que as que empregaram osso autógeno ou coágulo. Já em relação à MR, a média da variável obedeceu à relação: ACM > ALD = AU = CO (valores-p < 5%). Todas as intervenções apresentaram médias mais significativas de crescimento tecidual nas regiões mais próximas ao leito receptor. Foi possível concluir que os aloenxertos podem ser considerados soluções adequadas para o crescimento ósseo vertical. / Regarding different clinical applications for bone grafts in alveolar reconstructions and difficulties on achieving vertical osseous increase the present study performed a comparative histological evaluation of demineralized freeze-dried bone allograft (DFDBA), of fresh frozen bone allograft (FFBA), autogenous graft (AU) and blood clot (CO) on vertical guided bone regeneration (GBR) in rabbit calvarium. Nine rabbits were used, with one as the primary bone graft donor and eight that were subjected to a model of GBR, whereby 32 titanium cylinders were fixed to the calvaria and randomly filled with DFDBA, FFBA, AU, or CO. The animals were sacrificed 13 weeks later, and the content of the cylinders was subjected to hitomorphological and histomorphometric analysis to quantify the total area of neoformed tissue (AT), the new bone tissue (NB) and residual graft particles (RG). The results showed that mean values for AT were significant to DFDBA and FFBA and followed the relation DFDBA = FFBA > AU > CO. Considering new bone formation DFDBA and FFBA showed better results than the AU and CO. The amount of residual bone particles was larger in the DFDBA and followed the relation FFBA > DFDBA = AU = CO (pvalues < 5%). All interventions showed greater new tissue formation nearby the receptor site. It was possible to conclude that allografts DFDBA and FFBA can be considered good strategies for new bone formation in vertical increasing bone.

Dentre as diversas possibilidades de reconstrução de tecidos ósseos atróficos, a regeneração óssea guiada é uma das mais promissoras. Neste contexto, muitas membranas reabsorvíveis tem sido desenvolvidas e precisam ser testadas como parte de sua caracterização. O objetivo do presente estudo foi avaliar radiográfica e histologicamente em um estudo in vivo, se uma nova membrana polimérica eletrofiada de PCL/poli(rotaxona) demonstra comportamento semelhante a uma membrana de colágeno, comercialmente consagrada, quanto à promoção de regeneração óssea guiada. Foi realizado defeito crítico de 8mm de diâmetro na calvária de 60 ratos Wistar machos. Em dois grupos iguais (n=20) os defeitos foram recobertos aleatoriamente por uma membrana de colágeno suíno ou por uma membrana polimérica mista de policaprolactona (PCL) e poli(rotaxona). Em um terceiro grupo (n=20) os defeitos não foram recobertos e permaneceram apenas com o coágulo. Os animais sofreram eutanásia em 7, 14, 21 e 42 dias pós operatórios. Espécimes da região foram radiografadas e preparadas para análise histológica. Radiograficamente, os defeitos recobertos pela membrana de colágeno suíno apresentaram diminuição mais significativa da área radiográfica dos defeitos de acordo com a progressão dos períodos pós-operatórios do que nos outros grupos. A histomorfologia do reparo mostrou agrupamentos mais expressivos de células gigantes no grupo PCL/poli(rotaxona) sugerindo resposta à corpo estranho. Na histomorfometria, a neoformação óssea foi significativamente mais intensa e com osso neoformado mais maduro no grupo Colágeno. Concluímos que para um modelo de regeneração óssea guiada, a membrana de PCL/poli(rotaxona) não superou a membrana de colágeno. / The need to rebuild lost bone tissue, shows up as one of the great challenges of modern dentistry. Among several possibilities, guided bone regeneration is one of the most established techniques. In this context, many resorbable membranes have been developed and need to be tested as part of their characterization. The aim of this study was to evaluate by an in vivo model, if a new electrospinning PCL/polyrotaxane polymer membrane promotes similar guided bone regeneration when compared to a collagen membrane. An 8mm diameter critical defect was made in 60 male Wistar rats calvaria. In two equal groups (n = 20) the defects were randomly covered with a porcine collagen or a PCL/polyrotaxane membranes. In a third group (n = 20) the defects remained uncovered and just the blood clot occupied the defect. The animals were euthanized at 7, 14, 21 and 42 days post-operative. Specimens were x-rayed and prepared for histological analysis. Radiographically, the defects covered by porcine collagen membrane, showed more significant reduction in defect area, according to postoperative period evolution. Histomorphology showed intense giant cells presence in the PCL/polyrotaxane group, suggesting a foreign body response. The histomorphometric analysis showed new and mature bone formation more intense in collagen group. Under the limits of this study, the collagen membrane performance in guided tissue regeneration was far superior to the PCL/polyrotaxane membrane.

O desenvolvimento atual de materiais ósseo-substitutos com potencialidade de promover o fechamento completo de um defeito ósseo crítico tem levantado questões quanto à sua atuação biológica. Uma opção de material ósseo utilizado são os enxertos ósseos de origem animal, pois possuem propriedades físicoquímicas similares ao osso humano. O osso bovino misto que preserva a estrutura colagênica e o mineral ósseo tem sido proposto e utilizado como material ósseosubstituto. O objetivo desse estudo foi avaliar a biocompatibilidade e o potencial osteogênico de um enxerto bovino ósseo subistituto, OrthoGen® (Baumer S.A.), na forma de partículas (OGp) e blocos (OGb). Para a avaliação da biocompatibilidade, 100 mg de OrthoGen® nas formas de partícula e bloco, foram implantados no subcutâneo de ratos (n=25) e o tecido reacional foi avaliado aos 7, 14, 21, 30 e 60 dias (n=5 animais/período) após a implantação. Para a análise do potencial osteogênico foram implantados 100 mg de Orthogen® nas formas de partícula e bloco em defeito critico na calvária de ratos (n=30), e a formação óssea foi mensurada aos 1, 3 e 6 meses (n=10/período) após a implantação. A análise radiográfica e histomorfométrica revelaram que no tecido subcutâneo o OGb foi melhor aceito pelo organismo quando comparado ao OGp com uma frequência menor de células gigante multinucleadas entre os períodos avaliados (OGb 0,23% vs OGp 2,19%) e consequentemente uma média de reabsorção também menor (OGb 13% vs OGp 38%). Em ambos os implantes não foi encontrado focos de infiltrado inflamatório composto por leucócitos polimorfonucleares, linfócitos e plasmócitos. Na calvária o OGb mostrou níveis de reabsorção inferiores e uma maior taxa de formação óssea quando comparado ao OGp após 6 meses (OGb 70 mm³ vs OGp 17 mm³). Baseado no modelo experimental utilizado neste estudo, concluímos que ambas as formas do Orthogen® são biocompatíveis em tecido subcutâneo, no entanto, sua forma em bloco promove uma maior formação óssea, possuindo uma capacidade osteogênica superior à forma em partícula, no modelo experimental avaliado. / The current development of bone graft materials with the potential to promote the complete closure of a critical size bone defect has raised questions as to its biological activity. An option of bone material used, are animal bone grafts since the human bone have similar physicochemical properties. Among the materials is the integral bone substitute of bovine origin, which preserves the organic and inorganic compound of the bone tissue, has been proposed and used as bone graft. The aim of this study was to evaluate the biocompatibility and osteogenic potential of a new integral bone substitute OrthogenTM (Baumer S.A.) in the form of particles (OGp) and block (OGb). For biocompatibility evaluation, 100mg OrthogenTM was implanted into dorsal subcutaneous pocket of rat (n= 25) and the reactional tissue was analyzed at 7, 14, 21, 30 and 60 days (n=5animals/period) after implantation. For osteogenic potential evaluation, 100mg OrthogenTM was implanted into critical-size defect in parietal bones of rat (n=30) and the bone formation, biomaterial reabsorption, connective tissue formation and osteoclast activity was evaluated at 1, 3 and 6 months (n=10/period) after implantation. Radiographic and histomorphometrical analysis showed that, in the subcutaneous tissue the OGb was more accepted by the host compared to OGp, with lower density of the multinucleated giant cells (OGb 0.23% vs. OGp 2.19%) and consequently a lower rate of matrix resorption (OGb 13%, vs. OGp 38%). In both implants was not found focus of inflammatory infiltrated composed by polymorphonuclear leucocytes, lymphocytes and plasmocytes. In rat calvaria the OGb showed lower rate of reabsorption and more volume of bone formation compared to OGp after 6 months (OGb 70 mm3 vs OGp 17 mm3). Based on experimental models used in this study we concluded that both forms of the OrthogenTM was biocompatible in subcutaneous tissue, however, its form of porous block promoted greater bone formation and has a higher osteogenic capacity than the particle shape, in the evaluated experimental model.