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Bone Healing after implantation of bone substitute materials. Experimental studies in estrogen deficiency.Öberg, Sven January 2003 (has links)
<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>
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A Comparison of the Osteogenic Tissue Engineering Potential of Dental-Derived Stem Cell Lines: Stem Cells from Human Exfoliated Deciduous Teeth (SHEDs) vs. Periodontal Ligament Stem Cells (PERIOS)Vernon, Lauren Louise 01 January 2010 (has links)
The goal of this study is to assess the osteogenic potential of two types of dental stem cell lines within a tissue engineering application. More specifically, the goal of this study is to find a readily abundant cell source with capacity to express an osteogenic phenotype. There are two parameters utilized to evaluate tissue engineering potential of cells: proliferation rate and differentiation potential. Briefly, proliferation rate is the speed at which cells divide and differentiation potential determines if cells are capable of committing towards specific lineages (e.g. osteogenic). These components are important, because if cells are not expanding at a specific rate and are not differentiating towards the lineage desired, the tissue engineered will not mirror the characteristics of native tissue. Therefore, both components are necessary for osteogenic tissue engineering applications. Several stem cell lines have been isolated from different sources (e.g. umbilical, bone marrow) and characterized for their proliferative capacity and their potency. Among these progenitor or stem cell lines, are those isolated from human dental tissue. Due to the similarities between teeth and bone, this specific cell line may be useful in osteogenic tissue engineering applications. In this study, stem cells extracted from human exfoliated deciduous teeth (SHEDs) and periodontal ligament stem cells (PERIOs), were evaluated and compared. Briefly, to evaluate the proliferation rate an ex-vivo expansion study was conducted. This experiment found that both SHEDs and PERIOs were proliferative lines with doubling times of 23 hours and 19 hours respectively. Subsequently, osteogenic differentiation of SHEDs and PERIOs was assessed utilizing a 3-D fibrin gel suspension treated with osteogenic media containing either dexamethasone (DEX) or Retinoic Acid (RA) for 28 days. At day 28, osteogenic markers for collagen 1 (Col1), osteocalcin (OCN), and alkaline phosphatase (ALP) were evaluated using qPCR. Results demonstrated both SHEDs and PERIOs exhibited significant (p<0.05) increases in osteogenic gene expression under the influences of DEX and RA. However the most significant increases were expressed by the SHEDs that received the DEX treatment. Additionally, the synergistic ability of TGF-beta 3 on the osteogenic differentiation of the stem cells was evaluated. Cells were cultured in a 3-D fibrin gel suspension and allowed to differentiate in DEX osteogenic media with and without the supplementation of TGF-beta 3 for 21 days. Using qPCR the cells were evaluated for expression of Col1, OCN, and ALP. In both the SHEDs and PERIOs, the samples treated with TGF-beta 3 the osteogenic gene expression increased in reference to the control, but had a hindering effect compared to cells treated in DEX without the TGF-beta 3. These results from this study suggested, SHED cells grown in 3-D fibrin gel suspension, may be better than PERIO cells for osteogenic tissue engineering applications when treated with DEX media without the supplementation of TGF-beta 3.
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Πειραματική συγκριτική μελέτη αναγγείων μοσχευμάτων για την πλήρωση οστικών ελλειμάτων / Comparative experimental study of nonvascular bone grafts for bone defect fillingΑθανασίου, Βασίλειος 31 March 2010 (has links)
Σκοπός αυτής της πειραματικής μελέτης είναι ο έλεγχος βιολογικής συμπεριφοράς διαφόρων τύπων μοσχευμάτων που σήμερα χρησιμοποιούνται ευρέως ως υποκατάστατα οστοών.
Υλικό–Μέθοδος: Στην παρούσα μελέτη χρησιμοποιήθηκαν 90 κουνέλια Νέας Ζηλανδίας, ηλικία 3.5 μηνών και βάρους 4(0.25)kg, τα οποία χωρίσθηκαν σε 6 ομάδες, η κάθε μία εκ των οποίων περιελάμβανε 15 κουνέλια. Υπό γενική αναισθησία, με ενδομυϊκή χορήγηση κεταμίνης 35mg/kg και ξυλαζίνης 5mg/kg, δημιουργήθηκε, με πλάγια χειρουργική προσπέλαση, μια οπή με φρέζα διαμέτρου 4.5mm και βάθους 8mm, στους μηριαίους κονδύλους των 2 οπισθίων άκρων του κάθε κουνελιού (σύνολο 180 οπές). Στις οπές αυτές τοποθετήθηκαν τα ακόλουθα μοσχεύματα: Ομάδα Ι-αυτομόσχευμα, Ομάδα ΙΙ-αλλομόσχευμα (Grafton®), Ομάδα ΙΙΙ-ξενομόσχευμα (Lubboc®), Ομάδα ΙV-συνθετικό υποκατάστατο οστικών μοσχευμάτων (Ceraform®), Ομάδα V- συνθετικό υποκατάστατο οστικών μοσχευμάτων (Οsteoset®), Ομάδα VI-χωρίς μόσχευμα. Μετά την τοποθέτηση των μοσχευμάτων, τα κουνέλια θυσιάστηκαν με ενδοφλέβια νατριούχο θειοπεντάλη 5ml (pentothal) 10%, σε 1, 3 και 6 μήνες όπου έγινε λήψη δειγμάτων (το κάτω τριτημόριο του μηριαίου) για ιστολογική μελέτη. Τα δείγματα αξιολογήθηκαν με μια ιστολογική κλίμακα βαθμολόγησης 15-point για να καθοριστεί η ποιότητα της πώρωσης, η παρουσία οστικού ελλείμματος, η νέοαγγειογένεση και η αντιδραστική παρουσία κυττάρων φλεγμονής, καθώς και ο βαθμός ενσωμάτωσης και ανακατασκευής του πώρου.
Αποτελέσματα: Σύμφωνα με την ιστολογική κλίμακα το αυτομόσχευμα έδειξε τα καλύτερα αποτελέσματα σε όλες τις χρονικές στιγμές. Όλοι οι άλλοι τύποι μοσχεύματος έδειξαν σημαντικά κατώτερα αποτελέσματα σε σχέση με το αυτόλογο μόσχευμα (p≤0.05). Το Lubboc είχε σημαντικά καλύτερα αποτελέσματα σε σχέση με τα άλλα τρία μοσχεύματα (Grafton, Ceraform και Osteoset). Το Ceraform είχε τα κατώτερα αποτελέσματα σε όλες τις κατιγορίες
Συμπεράσματα: Το αυτόλογο μόσχευμα παραμένει το πρότυπο αναφοράς “gold standard” των μοσχευμάτων, επιδεικνύοντας εξαιρετικές ικανότητες ενσωμάτωσης. Το βόειο ξενομόσχευμα (Lubboc®) συνέβαλλε στη σύνθεση του πεταλιώδους οστού πιο αποτελεσματικά από το αλλομόσχευμα (Grafton®). Τα υποκατάστατα οστών (Ceraform® και Οsteoset®) ήταν κατώτερα από τα αλλομοσχεύματα και τα ξενομοσχεύματα / Background: Different types of bone-graft substitutes have been developed and are on the market worldwide to eliminate the drawbacks of autogenous grafting. This experimental animal study was undertaken to evaluate the different histological properties of various bone graft substitutes utilized in this hospital.
Material/Methods: Ninety New Zealand white rabbits were divided into six groups of 15 animals. Under general anesthesia, a 4.5 mm-wide hole was drilled into both the lateral femoral condyles of each rabbit, for a total of 180 condyles for analysis. The bone defects were filled with various grafts, these being 1) autograft, 2) DBM crunch allograft (Grafton(R)), 3) bovine cancellous bone xenograft (Lubboc(R)), 4) calcium phosphate hydroxyapatite substitute (Ceraform(R)), 5) calcium sulfate substitute (Osteoset(R)), and 6) no filling (control). The animals were sacrificed at 1, 3, and 6 months after implantation and tissue samples from the implanted areas were processed for histological evaluation. A histological grading scale was designed to determine the different histological parameters of bone healing.
Results: The highest histological grades were achieved with the use of cancellous bone autograft. Bovine xenograft (Lubboc) was the second best in the histological scale grading. The other substitutes (Grafton, Ceraform, Osteoset) had similar scores but were inferior to both allograft and xenograft.
Conclusions: Bovine xenograft showed better biological response than the other bone graft substitutes; however, more clinical studies are necessary to determine its overall effectiveness.
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Osso autógeno associado a osso bovino inorgânico (GenOx Inorg®) para aumento do soalho do seio maxilar e instalação de implantes: análise comparativa do potencial osteogênico de culturas de células derivadas do sítio doador e do sítio de implantação / The use of autogenous bone combined with anorganic bovine bone graft (GenOx Inorg®) for maxillary sinus augmentation and implat placement: a comparative analysis on the osteogenic potential of cell cultures derived from the donor site and the implant siteMelo, Willian Morais de 12 July 2012 (has links)
Objetivos: O objetivo desse estudo foi avaliar comparativamente o potencial osteogênico in vitro de células obtidas do ramo mandibular (RM, área doadora) e do seio maxilar enxertado com uma mistura de RM e osso bovino inorgânico (OBI), previamente à instalação de implantes de titânio (SM, sítio do seio maxilar enxertado). Material e Métodos: As células foram obtidas de três pacientes submetidos a procedimentos de aumento do soalho do seio maxilar com a proporção de 1:1 de RM e OBI (GenOx Inorg®). No momento da realização dos enxertos no seio maxilar e após 08 meses, antes da inserção dos implantes de titânio, fragmentos ósseos foram colhidos do RM e do SM, respectivamente, e submetidos à digestão enzimática com tripsina e colagenase para obtenção de células primárias. As células foram subcultivadas e crescidas sob condições osteogênicas por até 21 dias, tendo sido avaliados os seguintes parâmetros: proliferação/viabilidade celular, expressão gênica de marcadores osteoblásticos, atividade de fosfatase alcalina (ALP) e conteúdo de cálcio, por extração do vermelho de Alizarina. Culturas primárias derivadas do RM foram expostas ao GenOx Inorg® por 7 dias, quando se avaliou a atividade de ALP. Os resultados foram comparados por ANOVA two-way, seguido do teste de Tukey, ou pelo teste de Mann-Whitney. Resultados: Culturas do SM exibiram uma redução significante do potencial osteogênico se comparado ao de culturas do RM, com um aumento progressivo na proliferação celular associado a uma redução da expressão dos marcadores osteoblásticos, da atividade de ALP e do conteúdo de cálcio. A exposição do GenOx Inorg® às células primárias derivadas do RM inibiram a atividade de ALP. Conclusão: Esses resultados sugerem que o uso do GenOx Inorg® em associação a fragmentos do RM para aumento do soalho do seio maxilar inibe a diferenciação de células osteoblásticas no sítio de inserção de implantes de titânio após 8 meses de enxertia. / Objectives: This study aimed to comparatively evaluate the in vitro osteogenic potential of cells obtained from the mandibular ramus (MR, autogenous bone donor site) and from the maxillary sinus bone grafted with a mixture of anorganic bovine bone (ABB) and MR prior to titanium implant placement (MS, grafted implant site). Material and methods: Cells were obtained from three patients subjected to maxillary sinus floor augmentation with a 1:1 mixture of ABB (GenOx Inorg®) and MR. At the time of the sinus lift procedure and after 8 months, prior to implant placement, bone fragments were taken from MR and MS, respectively, and subjected to trypsin-collagenase digestion for primary cell culturing. Subcultured cells were grown under osteogenic condition for up 21 days and assayed for proliferation/viability, osteoblast marker mRNA levels, alkaline phosphatase (ALP) activity and calcium content/Alizarin red staining. ALP activity was also determined in primary explant cultures exposed to GenOx Inorg® (1:1 with MR) for 7 days. Data were compared using the two-way ANOVA followed by the Tukey test; otherwise, the Mann-Whitney test was used. Results: MS cultures exhibited a significantly lower osteogenic potential compared with MR cultures, with a progressive increase in cell proliferation together with a downregulation of osteoblast markers, reduced ALP activity and calcium content. Exposure of MR-derived primary cultures to GenOx Inorg® inhibited ALP activity. Conclusion: These results suggest that the use of GenOx Inorg® in combination with MR fragments for maxillary sinus floor augmentation inhibits the osteoblast cell differentiation at the implant site in the longterm.
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Prostorno određivanje položaja kalema u golenjači posle rekonstrukcije prednjeg ukrštenog ligamenta kolena / Determination of in-space position of tibial graft after reconstruction of anterior cruciate ligament of the kneeĐuričin Aleksandar 12 June 2018 (has links)
<p>Osnovni cilj rekonstrukcije prednjeg ukrštenog ligamenta kolena je dobijanje pune stabilnosti kolena u celom obimu pokreta. Bez obzira na razvoj operativne tehnike i rehabilitacije i dalje postoji jedan broj pacijenata koji nije u potpunosti zadovoljan rezultatom rekonstrukcije prednjeg ukrštenog ligamenta kolena, te je potrebno izvršiti ponovnu operaciju. Smatra se da je najčešći uzrok rane ponovne nestabilnosti loša pozicija kalema, tj. pozicija tunela koja nije na anatomskom mestu. Većina hirurga koji se bave ovom problematikom procenjuju poziciju kalema u golenjači na osnovu standardnih radiograma: prednje-zadnje i bočne projekcije, što svakako nije dovoljno precizno. U ekonomski razvijenim zemljama poziciju kalema određuju analizirajući snimke kompjuterizovane tomografije (CT) ili magnetne rezonance (MRI). Prvenstveno zbog smanjenja doze zračenja kojoj se izlažu pacijenti prilikom kompjuterizovane tomografije, ali i iz ekonomskih razloga bilo bi korisno razvijanje jednog lako dostupnog, jeftinog ali preciznog i jednostavnog metoda određivanja položaja kalema u golenjači. Osnovni cilj ovog istraživanja bio je da se dokaže značaj pozicije kalema u golenjači i mogućnost svakodnevne kliničke primene novorazvijenog kompjuterskog programa u cilju određivanja prostorne pozicije kalema u golenjači iz samo dva standardna radiograma. Studija je bila eksperimentalno-prospektivnog karaktera. Eksperimentalni deo istraživanja sproveden je na Fakultetu tehničkih nauka (FTN) u Novom Sadu na Departmanu za proizvodno mašinstvo gde je na osnovu standardnih RTG snimaka (prednje-zadnje i bočne projekcije) razvijen kompjuterski program za određivanje prostornog položaja kalema u golenjači. U cilju verifikacije novorazvijenog programa realizovano je prostorno određivanje položaja kalema u golenjači obradom standardnih RTG snimaka (prednje-zadnje i bočne projekcije) kod 10 pacijenata kod kojih je izvršena primarna rekonstrukcija prednjeg ukrštenog ligamenta kolena. Dobijeni rezultati su upoređeni sa položajem kalema na CT snimcima koji su obezbeđeni za sve pacijente. Na taj način je eksperimentalnim putem utvrđena preciznost novorazvijenog softvera u određivanju prostornog položaja kalema u golenjači. Drugi deo istraživanja bilo je prospektivno kliničko ispitivanje koje je sprovedeno na Klinici za ortopedsku hirurgiju i traumatologiju Kliničkog centra Vojvodine u Novom Sadu. Ispitivanu grupu je činilo 120 pacijenata, oba pola, sa prekidom prednjeg ukrštenog ligamenta levog ili desnog kolena. Metodom slučajnog izbora pacijenti su podeljeni u četiri grupe od po 30 pacijenata prema veličini sagitalnog ugla (S) bušenja kanala u golenjači (S 60º-69,9º i S 70º-80º) i prema veličini transverzalnog ugla (T) bušenja kanala u golenjači (T 60º-69,9º i T 70º-80º). Grupa I 30 pacijenata (S 60º-69,9º i T 60º-69,9º), grupa II 30 pacijenata (S 60º-69,9º i T 70º-80º), grupa III 30 pacijenata (S 70º-80º i T 60º-69,9º), grupa IV 30 pacijenata (S 70º-80º i T 70º-80º). Svi pacijenti su godinu dana nakon operacije prošli klinička ispitivanja po bodovnim skalama (Tegner bodovna skala, Lysholm bodovna skala i IKDC standard) i artrometrijska merenja. Rezultati dobijeni merenjem položaja kalema u golenjači, kliničkim ispitivanjima i artrometrijskim merenjima poređani su unutar svake grupe posebno, a izvršeno je i poređenje dobijenih rezultata između svih grupa. U ispitivanje su uključeni samo oni pacijenti koji su dali potpisani informisani pristanak da učestvuju u ispitivanju nakon detaljnog upoznavanja sa planiranom procedurom. Svaki pacijent je bio informisan o svrsi i načinu sprovođenja istraživanja, kao i o pregledima i merenjima koja će biti vršena. Statističkom analizom rezultata utvrđeno je da veličina greške prostornog određivanja položaja kalema u golenjači posle rekonstrukcije prednjeg ukrštenog ligamenta kolena novorazvijenim kompjuterskim programom nije statistički značajna, a kompjutersko određivanje položaja kalema u golenjači omogućava iste rezultate kao i CT snimci. Sumiranjem zaključaka nakon sveobuhvatne analize dobijenih rezultata istraživanja, može se zaključiti da pozicija kalema u golenjači posle rekonstrukcije prednjeg ukrštenog ligamenta kolena utiče na postoperativni funkcionalni rezultat.</p> / <p>The main goal of reconstruction of the anterior cruciate ligament of the knee is to obtain complete knee stability in the full range of movement. Regardless of the development of operational techniques and rehabilitation, there is still a number of patients who are not completely satisfied with the result of reconstruction of the anterior cruciate ligament, and a re-operation is required. It is believed that the most common cause of the instability is the bad position of the graft, i.e. position of the tunnel that is not at the anatomical place insertion. Most surgeons who deal with this problem evaluate the position of the graft in the tibia based on standard radiograms: anterior-posterior and lateral projections, which is not precise enough. In economically developed countries, the position of the graft is determined by analyzing images of computerized tomography (CT) or magnetic resonance (MRI). Primarily due to a decrease in the radiation dose exposed to patients during computerized tomography, but also for economic reasons, it would be useful to develop an easily accessible, inexpensive but precise and simple method for determining the position of the graft in the tibia. The main goal of this examination was to prove the significance of the position of the graft and the possibility of daily clinical use of the newly developed computer program in order to determine in-space position of the graft in only two standard radiograms. The study was experimental-prospective. The experimental part of the research was conducted at the Faculty of Technical Sciences (FTN) in Novi Sad at the Department of Production Engineering, where a computer program for determining in-space position of the graft in the tibia was developed on the basis of standard RTG images (anterior-posterior and lateral projections). In order to verify the newly developed program, in-space determination of the position of the graft in the tibia by processing standard RTG images (anterior-posterior and lateral projections) was performed in 10 patients in which the primary reconstruction of the anterior cruciate ligament was performed. The obtained results were compared with the position of the grafts on CT images provided to all patients. In this way, the accuracy of newly developed software in determining in-space position of the graft in the tibia was determined experimentally. The second part of the study was a prospective clinical trial conducted at the Clinic for Orthopedic Surgery and Traumatology at the Clinical Center of Vojvodina in Novi Sad. The investigated group consisted of 120 patients, both sexes, with a break of the anterior cruciate ligament of the left or right knee. By random selection, patients were divided into four groups of 30 patients according to the size of the sagital angle (S) of the tunnel drilling in the tibia (S 60º-69,9º and S 70º-80º) and according to the transversal angle (T) of the drill tunnel in the tibia ( T 60º-69.9º and T 70º-80º). Group I 30 patients (S 60º-69.9º and T 60º-69.9º), group II 30 patients (S 60º-69.9º and T 70º-80º), group III 30 patients (S 70º-80º and Tº60º -69.9º), group IV 30 patients (S 70º-80º and T 70º-80º). All of the patients underwent clinical trials by scales (Tegner score scale, Lysholm score scale and IKDC standard) and arthrometric measurements one year after surgery. The results obtained by measuring the position of the graft in the tibia, clinical trials and arthrometric measurements were classified separately in each group, and obtained results were compared between all groups. The study included only those patients who gave signed informed consent to participate in the study after being thoroughly informed about planned procedure. Each patient was informed about the purpose and method of conducting the research, as well as the examinations and measurements to be performed. Statistical analysis of the results showed that the size of the error in measuring in-space determination of the position of the graft in tibia after reconstruction of the anterior cruciate ligament of the knee by a newly developed computer program was not statistically significant, and the computer determination of the position of the graft in the tibia provides the same results as the CT images. Summarizing the conclusions after a comprehensive analysis of the obtained results of the study, it can be concluded that the position of the graft in tibia after reconstruction of the anterior cruciate ligament affects the postoperative functional result.</p>
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Osso autógeno associado a osso bovino inorgânico (GenOx Inorg®) para aumento do soalho do seio maxilar e instalação de implantes: análise comparativa do potencial osteogênico de culturas de células derivadas do sítio doador e do sítio de implantação / The use of autogenous bone combined with anorganic bovine bone graft (GenOx Inorg®) for maxillary sinus augmentation and implat placement: a comparative analysis on the osteogenic potential of cell cultures derived from the donor site and the implant siteWillian Morais de Melo 12 July 2012 (has links)
Objetivos: O objetivo desse estudo foi avaliar comparativamente o potencial osteogênico in vitro de células obtidas do ramo mandibular (RM, área doadora) e do seio maxilar enxertado com uma mistura de RM e osso bovino inorgânico (OBI), previamente à instalação de implantes de titânio (SM, sítio do seio maxilar enxertado). Material e Métodos: As células foram obtidas de três pacientes submetidos a procedimentos de aumento do soalho do seio maxilar com a proporção de 1:1 de RM e OBI (GenOx Inorg®). No momento da realização dos enxertos no seio maxilar e após 08 meses, antes da inserção dos implantes de titânio, fragmentos ósseos foram colhidos do RM e do SM, respectivamente, e submetidos à digestão enzimática com tripsina e colagenase para obtenção de células primárias. As células foram subcultivadas e crescidas sob condições osteogênicas por até 21 dias, tendo sido avaliados os seguintes parâmetros: proliferação/viabilidade celular, expressão gênica de marcadores osteoblásticos, atividade de fosfatase alcalina (ALP) e conteúdo de cálcio, por extração do vermelho de Alizarina. Culturas primárias derivadas do RM foram expostas ao GenOx Inorg® por 7 dias, quando se avaliou a atividade de ALP. Os resultados foram comparados por ANOVA two-way, seguido do teste de Tukey, ou pelo teste de Mann-Whitney. Resultados: Culturas do SM exibiram uma redução significante do potencial osteogênico se comparado ao de culturas do RM, com um aumento progressivo na proliferação celular associado a uma redução da expressão dos marcadores osteoblásticos, da atividade de ALP e do conteúdo de cálcio. A exposição do GenOx Inorg® às células primárias derivadas do RM inibiram a atividade de ALP. Conclusão: Esses resultados sugerem que o uso do GenOx Inorg® em associação a fragmentos do RM para aumento do soalho do seio maxilar inibe a diferenciação de células osteoblásticas no sítio de inserção de implantes de titânio após 8 meses de enxertia. / Objectives: This study aimed to comparatively evaluate the in vitro osteogenic potential of cells obtained from the mandibular ramus (MR, autogenous bone donor site) and from the maxillary sinus bone grafted with a mixture of anorganic bovine bone (ABB) and MR prior to titanium implant placement (MS, grafted implant site). Material and methods: Cells were obtained from three patients subjected to maxillary sinus floor augmentation with a 1:1 mixture of ABB (GenOx Inorg®) and MR. At the time of the sinus lift procedure and after 8 months, prior to implant placement, bone fragments were taken from MR and MS, respectively, and subjected to trypsin-collagenase digestion for primary cell culturing. Subcultured cells were grown under osteogenic condition for up 21 days and assayed for proliferation/viability, osteoblast marker mRNA levels, alkaline phosphatase (ALP) activity and calcium content/Alizarin red staining. ALP activity was also determined in primary explant cultures exposed to GenOx Inorg® (1:1 with MR) for 7 days. Data were compared using the two-way ANOVA followed by the Tukey test; otherwise, the Mann-Whitney test was used. Results: MS cultures exhibited a significantly lower osteogenic potential compared with MR cultures, with a progressive increase in cell proliferation together with a downregulation of osteoblast markers, reduced ALP activity and calcium content. Exposure of MR-derived primary cultures to GenOx Inorg® inhibited ALP activity. Conclusion: These results suggest that the use of GenOx Inorg® in combination with MR fragments for maxillary sinus floor augmentation inhibits the osteoblast cell differentiation at the implant site in the longterm.
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Engineering Bioactive And Multifunctional Graphene Polymer Composites for Bone Tissue RegenerationKumar, Sachin B January 2016 (has links) (PDF)
The growing incidences of orthopedic problems globally have created a huge demand for strong bioactive materials for bone tissue engineering. Over the years, studies have shown chemical, physical, and mechanical properties of biomaterials influence the cellular interactions at the material-tissue interface, which subsequently controls biological response to materials. Strong biomaterials with surface properties that actively direct cellular response hold the key for engineering the next generation orthopedic implants. With its unique properties graphene can be used to reinforce poly (ε-caprolactone) (PCL) to prepare strong and bioactive polymer nanocomposites for bone tissue regeneration. The thesis entitled ―Engineering bioactive and multifunctional graphene polymer composites for bone tissue regeneration” systematically studies the effect of different chemically functionalized and metal-graphene hybrid nanoparticles in PCL composites for bone tissue engineering. The thesis comprises of seven chapters. Chapter 1 is an outline review on the impact of graphene and graphene derived particles to prepare supporting substrates for tissue regeneration and the associated cell response to multifunctional graphene substrate. This chapter discusses how cells interact with different graphene based particles and the interplay between cells performance and multifunctional properties of graphene based substrates.
Chapter 2 describes the role, if any, of the functionalization of graphene on mechanical properties, stem cell response and bacterial biofilm formation. PCL composites of graphene oxide (GO), reduced GO (RGO) and amine-functionalized GO (AGO) were prepared at different filler contents (1%, 3% and 5%). Although the addition of the nanoparticles to PCL markedly increased the storage modulus, this increase was higher for GO and AGO than with RGO. In vitro cell studies revealed that the AGO and GO particles significantly increased human mesenchymal stem cell (hMSC) proliferation. AGO was most effective in augmenting stem cell osteogenesis leading to mineralization. Bacterial studies revealed that interaction with functionalized GO induced bacterial cell death due to membrane damage which was further accentuated by amine groups in AGO. The synergistic effect of oxygen containing functional groups and amine groups on AGO-reinforced composites renders the optimal combination of improved modulus, favorable stem cell response and biofilm inhibition desired for orthopaedic applications. In Chapter 3, toward preparing strong multi-biofunctional materials, poly(ethylenimine) (PEI) conjugated graphene oxide (GO_PEI) was synthesized using poly(acrylic acid) (PAA) as spacer and incorporated in PCL at different fractions. GO_PEI significantly promoted proliferation and formation of focal adhesions in hMSCs on PCL. GO_PEI was highly potent in inducing stem cell osteogenesis leading to 90% increase in alkaline phosphatase activity and mineralization over neat PCL with 5% filler content and was 50% better than GO. Remarkably, 5% GO_PEI was as potent as soluble osteo-inductive factors. Increased adsorption of osteogenic factors due to the amine and oxygen containing functional groups on GO_PEI augment stem cell differentiation. GO_PEI was also highly efficient in imparting bactericidal activity with 85% reduction in counts of E. coli colonies compared to neat PCL at 5% filler content and was more than twice as efficient as GO. This may be attributed to the synergistic effect of the sharp edges of the particles along with the presence of the different chemical moieties. Thus, in contrast to using labile biomolecules, GO_PEI based polymer composites can be utilized to prepare bioactive resorbable biomaterials for fabricating orthopedic devices for fracture fixation and tissue engineering.
Chapter 4 describes the preparation of hybrid nanoparticles of graphene sheets decorated with strontium metallic nanoparticles and its advantages in bone tissue engineering. Strontium-decorated reduced graphene oxide (RGO_Sr) nanoparticles were synthesized by
facile reduction of graphene oxide and strontium nitrate. X-ray diffraction, transmission electron microscopy, and atomic force microscopy revealed that the hybrid particles were composed of RGO sheets decorated with 200 – 300 nm metallic strontium particles. Thermal gravimetric analysis further confirmed the composition of the hybrid particles as 22 wt% of strontium. Macroporous tissue scaffolds were prepared incorporating RGO_Sr particles in PCL. The PCL/RGO_Sr scaffolds were found to elute strontium ions in aqueous medium. Osteoblast proliferation and differentiation was significantly higher in the PCL scaffolds containing the RGO_Sr particles in contrast to neat PCL and PCL/RGO scaffolds. The increased biological activity can be attributed to the release of strontium ions from the hybrid nanoparticles. This study demonstrates that composites prepared using hybrid nanoparticles that elute strontium ions can be used to prepare scaffolds with osteoinductive property. These findings have important implications for designing the next generation of biomaterials for use in tissue regeneration.
Chapter 5 discusses the use of hybrid graphene-silver particles (RGO_Ag) to reinforce PCL and compared with PCL/RGO and PCL/Ag composites containing RGO and silver nanoparticles (AgNPs), respectively. RGO_Ag hybrid particles were well dispersed in the PCL matrix unlike the RGO and AgNPs due to enhanced exfoliation. RGO_Ag led to 77 % increase in the modulus of PCL and provided a conductive network for electron transfer. Electrical conductivity increased four orders of magnitude from 10-11 S/cm to 10-7 S/cm at 5 wt % filler that greatly exceeded the improvements with the use of RGO and AgNP in PCL. RGO_Ag particles reinforced in PCL showed sustained release of silver ions from the PCL matrix unlike the burst release from PCL/Ag. PCL/RGO_Ag and PCL/RGO composites were non-toxic to hMSCs and supported osteogenic differentiation unlike the PCL/Ag composites which were highly toxic at ≥3% filler content. The PCL/RGO_Ag composites exhibited good antibacterial effect due to a combination of silver ion release from the AgNPs and the mechanical rupture induced by the RGO in the hybrid nanoparticles. Thus, the synergistic effect of Ag and RGO in the PCL matrix uniquely yielded a multifunctional material for use in implantable biomedical devices and tissue engineering. Chapter 6 presents investigation of potential differences in the biological response to graphene in polymer composites in the form of 2D substrates and 3D scaffolds. Results showed that osteoblast response to graphene in polymer nanocomposites is markedly altered between 2D substrates and 3D scaffold due to the roughness induced by the sharp edges of graphene at the surface in 3D but not in 2D. Osteoblast organized into aggregates in 3D scaffolds in contrast to more well spread and randomly distributed cells on 2D discs due to the macro-porous architecture of the scaffolds. Increased cell-cell contact and altered cellular morphology led to significantly higher mineralization in 3D scaffolds compared to 2D. This study demonstrates that the cellular response to nanoparticles in composites can change markedly by varying the processing route.
Chapter 7 summarizes the important results and future directions of the work. This chapter provides general conclusions arising from this study, and makes suggestions for future work designed to provide a greater understanding of the in vivo response in terms of bio-distribution of the released functionalized graphene from the scaffold or substrate must be assessed with special attention on their accumulation or excretion.
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