6 research outputs found
Effects of platelet-rich fibrin produced by three centrifugation protocols on bone neoformation in defects created in rat calvaria.
This study evaluated the potential of Leukocyte-platelet-rich fibrin (L-PRF; fixed angle centrifugation protocol), Advanced-platelet-rich fibrin (A-PRF; low-speed fixed angle centrifugation protocol), and Horizontal-platelet-rich fibrin (H-PRF; horizontal centrifugation protocol) in bone neoformation in critical size defects (CSDs) in rat calvaria. Thirty-two rats were divided into groups: Control (C), L-PRF, A-PRF, and H-PRF. 5 mm diameter CSDs were created in the animals' calvaria. Defects from group Control (C) were filled with blood clots, while defects from groups L-PRF, A-PRF, and H-PRF were filled with respective platelet-rich fibrin (PRF) membranes. L-PRF, A-PRF, and H-PRF were prepared from animal blood collection and specific centrifugation protocols. At 14 and 30 days, calcein (CA) and alizarin (AL) injections were performed, respectively. Animals were euthanized at 35 days. Microtomographic, laser confocal microscopy, and histomorphometric analyzes were performed. Data were statistically analyzed (ANOVA, Tukey, p < .05). L-PRF, A-PRF, and H-PRF groups showed higher values of bone volume (BV), newly formed bone area (NFBA), and precipitation of CA and AL than the C group (p < .05). The H-PRF group showed higher values of BV, number of trabeculae (Tb. N), NFBA, and higher precipitation of AL than the A-PRF and L-PRF groups (p < .05). Therefore, it can be concluded that: i) L-PRF, A-PRF, and H-PRF potentiate bone neoformation in CSDs in rat calvaria; ii) H-PRF demonstrated more biological potential for bone healing
Effect of 3D scaffolds funcionalized with anti-fibronectin aptamers on blood clot formation and osteogenesis. Ex vivo and in vivo study in rats
Na odontologia, o uso de arcabouços tridimensionais tem sido empregado na regeneração óssea maxilo-facial. A adsorção de moléculas bioativas sobre a superfície de arcabouços (SCA) possivelmente otimize a osteogênese. Assim, nessa pesquisa avaliou ex vivo, in vitro (osteoblastos - OSB) e in vivo (defeito em calvária em 5 e 15 dias) o efeito da funcionalização de SCA (quitosana +20% de β-TCP) com aptâmeros (APT) anti-fibronectina (anti-FN) na osteogênese. A funcionalização dos SCA foi realizada com 20 µg de APT por simples adsorção. O coágulo fisiológico (PhC) foi desenvolvido por 16 horas sobre os SCA com ou sem aptamêros (APT) em defeitos críticos criados em calvária de ratos, caracterizando o estudo ex vivo. Em seguida, os OSB foram cultivados sobre o PhC formado nos SCA com e sem APT. Os ensaios in vitro foram realizados nos SCA sem PhC. A morfologia dos OSB e do PhC foi verificada por MEV. A caracterização celular do PhC foi realizada por citometria de fluxo (CF). A viabilidade celular, acúmulo de matriz mineralizada e expressão de ALP e BSP foram investigadas por MTT, ARS e IF, respectivamente. Adicionalmente, a expressão dos genes Alp, Runx2, Bsp, Ocn e Tgf-β1 foi verificada por RT-PCR. No experimento in vivo, foram realizadas as análises de Micro-CT, morfometria por coloração de tricrômio de Masom, IF, análise transcriptômica por RNAseq e RT-PCR em 5 e 15 dias nos grupos SCA e SCA+APT. Os resultados ex vivo mostraram elevada imunomarcação da ALP e IBSP no grupo com APT. A CF revelou maior detecção de CD90, CD45 e CD44 no PhC formado no mesmo grupo. Na MEV foi observada uma densa rede de fibrina enriquecida, composta com diferentes tipos celulares no PhC do SCA com APT. O RT-PCR mostrou mais expressão do gene Tgf-β1 no grupo SCA+APT+PhC+OSB. Em relação aos ensaios in vitro (sem o PhC) houve mais formação de matriz mineralizada em SCA+OSB+APT em 10 dias. O mesmo foi observado na IF evidenciando mais imunomarcação da IBSP em SCA+APT+OSB, enquanto a ALP foi mais prevalente no grupo com SCA+OSB. Nas análises in vivo, o Micro-CT revelou maior volume ósseo e menos porosidade óssea em 5 dias em SCA+APT. Além disso, houve mais formação de tecido colágeno, alguns pontos de formação óssea e elevada expressão da ALP no grupo APT em 5 dias, enquanto a expressão de RUNX2 foi mais predominante em SCA+APT 15 dias. Houveram mais genes diferencialmente expressos em 15 dias, segundo o RNAseq. A ontologia gênica (GO), mostrou diferenças nas funções biológicas associadas à adesão celular e aos canais iônicos da membrana celular. Em 15 dias, essa diferença foi associada à resposta imune, aos componentes da MEC, da atividade antioxidante e da polimerização do citoesqueleto celular (regulada negativamente). Esses achados foram validados verificando a expressão dos genes Vim, Hprt1, Clcn4, Cd24, Krtap7-1, Psme2, Tnf e Il-1β. Em conjunto, os resultados mostraram que o APT proporciona melhora no padrão de coagulação, na diferenciação osteoblástica e nos eventos iniciais associados à osteoimunologia.In dentistry, the use of three-dimensional scaffolds (SCA) has been used in maxillofacial bone regeneration. The adsorption of bioactive molecules on SCA surface possibly optimizes osteogenesis. Thus, in this research were evaluated, ex vivo, in vitro (osteoblasts - OSB) and in vivo (calvaria defect at 5 and 15 days) the effect of SCA functionalization (chitosan +20% β-TCP) with anti-fibronectin aptamers (APT) on osteogenesis. SCA functionalization was performed with 20 µg of APT by simple adsorption. The physiological coagulum (PhC) was developed for 16 hours on the SCA with or without APT in critical defects created in rat calvaria, characterizing the ex vivo study. Then, the OSB were cultured on the PhC formed on SCA with and without APT. In vitro assays were performed on SCAs without PhC. The OSB and PhC morphology was verified by SEM. The cellular characterization of PhC was performed by flow cytometry (CF). Cell viability, mineralized matrix accumulation and ALP and BSP expression were investigated by MTT, ARS and IF, respectively. Additionally, the Alpl, Runx2, Bsp, Ocn and Tgf-β1 gene expression was verified by RT-PCR. In the in vivo experiment, Micro-CT, morphometry by Masom\'s trichrome staining, IF, transcriptomic analysis by RNAseq and RT-PCR were performed at 5 and 15 days in the SCA and SCA+APT groups. The ex vivo results showed high ALP and IBSP labelind in the APT group. FC revealed greater detection of CD90, CD45 and CD44 in PhC formed in the same group. In SEM, a dense fibrina enriched network was observed, composed of different cell types in PhC formed on SCA with APT. RT-PCR showed more Tgf-β1 gene expression in the SCA+APT+PhC+OSB group. In relation to in vitro assays (without the PhC) there was more formation of mineralized matrix in SCA+APT+OSB group in 10 days. The same was observed in IF, showing more immunostaining of IBSP in SCA+APT+OSB, while ALP was more prevalent in the SCA+OSB group. In in vivo analyses, Micro-CT revealed greater bone volume and less bone porosity at 5 days in SCA+APT. In addition, there was more collagen tissue formation, some bone formation points and increased ALP expression in the APT group at 5 days, while RUNX2 expression was more predominant in SCA+APT at 15 days. In RNAseq showed more differentially expressed genes in 15 days. Gene ontology (GO) showed differences in biological functions associated with cell adhesion and cell membrane ion channels. At 15 days, this difference was associated with the immune response, ECM components, antioxidant activity and cellular cytoskeleton polymerization (downregulated). These findings were validated by verifying Vim, Hprt1, Clcn4, Cd24, Krtap7-1, Psme2, Tnf and Il-1β gene expression. Taken together, these results showed that APT improves the coagulation pattern, osteoblastic differentiation and early events associated with osteoimmunology
Effects of exposure to aluminum citrate in a model of induced alveolar bone loss in rats
282-287Aluminum (Al) is the most abundant metal in the earth's crust and is found naturally in the air, water, and soil. Al may pose a major threat to humans, causing diseases and bone disorders. Nevertheless, the effects of Al on alveolar bone are not reported in the literature. Here, we investigated the effects of sub-chronic intoxication with Al citrate in a model of alveolar bone loss, induced by ligature, in male Wistar rats. The animals were exposed to Al citrate (100 mg/kg/day), by gavage, for 30 days. We used 40 rats equally divided into four groups: Control; Experimental Periodontitis (EPeriodontitis); Al; Al+EPeriodontitis. After exposure period, we evaluated the concentration of Al in the blood and alveolar bone, as well as the Al measurement in alveolar bone. The results showed that the Al exposure promoted distribution of Al in blood serum and in the alveolar bone. The Al exposure per si results in alveolar bone loss as well as potentiates damages of periodontitis induced cases. These results suggest that levels aluminum in the blood and alveolar bone induce alveolar bone loss, besides present aggravating effect in the presence of induced periodontitis
Anti-Fibronectin Aptamer Modifies Blood Clot Pattern and Stimulates Osteogenesis: An Ex Vivo Study.
BACKGROUND
Scaffold (SCA) functionalization with aptamers (APT) provides adsorption of specific bioactive molecules on biomaterial surfaces. The aim of this study was to observe if SCA enriched with anti-fibronectin APT can favor coagulum (PhC) and osteoblasts (OSB) differentiation.
METHODS
20 μg of APT was functionalized on SCA by simple adsorption. For PhC formation, SCAs were inserted into rat calvaria defects for 17 h. Following proper transportation (buffer solution PB), OSBs (UMR-106 lineage) were seeded over PhC + SCAs with and without APT. Cells and PhC morphology, PhC cell population, protein labeling and gene expression were observed in different time points.
RESULTS
The APT induced higher alkaline phosphatase and bone sialoprotein immunolabeling in OSB. Mesenchymal stem cells, leukocytes and lymphocytes cells were detected more in the APT group than when scaffolds were not functionalized. Additionally, an enriched and dense fibrin network and different cell types were observed, with more OSB and white blood cells in PhC formed on SCA with APT. The gene expression showed higher transforming growth factor beta 1 (TGF-b1) detection in SCA with APT.
CONCLUSIONS
The SCA functionalization with fibronectin aptamers may alter key morphological and functional features of blood clot formation, and provides a selective expression of proteins related to osteo differentiation. Additionally, aptamers increase TGF-b1 gene expression, which is highly associated with improvements in regenerative therapies
Comparison of the effects of platelet concentrates produced by high and low-speed centrifugation protocols on the healing of critical-size defects in rat calvaria: a microtomographic and histomorphometric study.
The current study evaluated the healing of critical-size defects (CSD) created in rat calvaria treated with platelet concentrates produced by high-speed (Leukocyte- and Platelet-Rich Fibrin - L-PRF) and low-speed (Advanced Platelet-Rich Fibrin - A-PRF) protocols of centrifugation. Twenty-four rats were distributed into three groups: Control, L-PRF, and A-PRF. Five mm diameter CSD were created on the animals' calvaria. The defects of the L-PRF and A-PRF groups were filled with 0.01 ml of L-PRF and A-PRF, respectively. The control group defects were filled with a blood clot only. All animals were euthanized on the 35th postoperative day. Histomorphometric and microtomographic analyses were then performed. The L-PRF and A-PRF groups had significantly higher bone volume and neoformed bone area than those of the control group and lowered bone porosity values (p < .05). No significant differences were observed between A-PRF and L-PRF groups for the analyzed parameters. Therefore, it can be concluded that i) L-PRF and A-PRF potentiated the healing of CSD in rat calvaria; ii) high and low-speed centrifugation protocols did not produce PRF matrices with different biological impacts on the amount of bone neoformation