121 results on '"Beloti MM"'
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2. Mandibular symphysis and ramus as sources of osteoblastic cells for bone tissue engineering.
- Author
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Ferraz, EP, Xavier, SP, Oliveira, PT, Beloti, MM, and Rosa, AL
- Subjects
ALKALINE phosphatase ,ANALYSIS of variance ,AUTOGRAFTS ,BIOMEDICAL engineering ,BONES ,GENE expression ,MANDIBLE ,RESEARCH methodology ,POLYMERASE chain reaction ,STATISTICS ,TISSUE culture ,PHENOTYPES ,DATA analysis - Abstract
Objectives Autografts from mandibular symphysis and ramus are often used for bone reconstruction. Based on this, we hypothesized that these sites could be useful cell sources for bone tissue engineering approaches. Thus, our study aimed at evaluating the proliferation and osteoblast phenotype development of cells derived from mandibular symphysis and ramus. Materials and Methods Cells were isolated from bone fragments of four patients by enzymatic digestion and cultured under osteogenic condition for up to 17 days. Cultures were assayed for cell proliferation, gene expression of key bone markers runt-related transcription factor 2 (Runx2), distal-less homeobox 5 ( DLX5), SATB homeobox 2 ( SATB2), Osterix ( OSX), family with sequence similarity 20, member C ( FAM20C), bone sialoprotein ( BSP), osteopontin ( OPN) and osteocalcin ( OC), alkaline phosphatase ( ALP) expression and activity, and extracellular matrix mineralization. Data were compared by two-way ANOVA or t-test for independent samples when appropriate. Results Cells derived from ramus displayed lower proliferative activity and higher gene expression of Runx2, DLX5, SATB2, OSX, FAM20C, BSP, OPN and OC, ALP protein expression and activity and extracellular matrix mineralization compared with symphysis-derived cells. Conclusion Symphysis and ramus may be considered as cell sources for bone tissue engineering approaches but due to the higher osteogenic potential, ramus-derived cells are more appealing for constructing cell-based biomaterials. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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3. Osseointegration of porous hydroxy apatite implanted in rabbits
- Author
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Beloti, Mm, Oliveira, Pt, Noort, R., and Adalberto Luiz Rosa
4. Effect of cpTi and Ti6Al4V on rat bone narrow cells
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Beloti, Mm, Fidelis, Gt, and Adalberto Luiz Rosa
5. Nitric acid passivation does not affect in vitro biocompatibility of titanium
- Author
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Faria, Acl, Beloti, Mm, and Adalberto Luiz Rosa
6. Jaboticaba Peel Extract Attenuates Ovariectomy-Induced Bone Loss by Preserving Osteoblast Activity.
- Author
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Adolpho LF, Gomes MPO, Freitas GP, Bighetti-Trevisan RL, Ramos JIR, Campeoti GH, Zatta GC, Almeida ALG, Tarone AG, Marostica-Junior MR, Rosa AL, and Beloti MM
- Abstract
Therapies to prevent osteoporosis are relevant since it is one of the most common non-communicable human diseases in the world and the most prevalent bone disorder in adults. Since jaboticaba peel extract (JPE) added to the culture medium enhanced the osteogenic potential of mesenchymal stem cells (MSCs) derived from osteoporotic rats, we hypothesized that JPE prevents the development of ovariectomy-induced osteoporosis. Ovariectomized rats were treated with either JPE (30 mg/kg of body weight) or its vehicle for 90 days, starting 7 days after the ovariectomy. Then, the femurs were subjected to microcomputed tomography and histological analyses, and the osteoblast and adipocyte differentiation of MSCs was evaluated. JPE attenuated ovariectomy-induced bone loss, as evidenced by higher bone volume/total volume and trabecular number, along with lower trabecular separation and bone marrow adiposity. These protective effects of JPE on bone tissue are due to its ability to prevent the imbalance between osteoblast and adipocyte differentiation of MSCs, since, compared with MSCs derived from ovariectomized rats treated with vehicle, MSCs treated with JPE exhibited higher gene and protein expression of osteogenic markers and extracellular matrix mineralization, as well as lower gene expression of adipogenic markers. These data highlight the potential therapeutic use of JPE to prevent osteoporosis.
- Published
- 2024
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7. Efficacy of mesenchymal stem cell-based therapy on the bone repair of hypertensive rats.
- Author
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Souza ATP, Freitas GP, Lopes HB, Weffort D, Adolpho LF, Gomes MPO, Oliveira FS, Almeida ALG, Beloti MM, and Rosa AL
- Abstract
Objective: Hypertension disrupts the bone integrity and its repair ability. This study explores the efficiency of a therapy based on the application of mesenchymal stem cells (MSCs) to repair bone defects of spontaneously hypertensive rats (SHR)., Methods: First, we evaluated SHR in terms of bone morphometry and differentiation of MSCs into osteoblasts. Then, the effects of the interactions between MSCs from normotensive rats (NTR-MSCs) cocultured with SHR (SHR-MSCs) on the osteoblast differentiation of both cell populations were evaluated. Also, bone formation into calvarial defects of SHR treated with NTR-MSCs was analyzed., Results: Hypertension induced bone loss evidenced by reduced bone morphometric parameters of femurs of SHR compared with NTR as well as decreased osteoblast differentiation of SHR-MSCs compared with NTR-MSCs. NTR-MSCs partially restored the capacity of SHR-MSCs to differentiate into osteoblasts, while SHR-MSCs exhibited a slight negative effect on NTR-MSCs. An enhanced bone repair was observed in defects treated with NTR-MSCs compared with control, stressing this cell therapy efficacy even in bones damaged by hypertension., Conclusion: The use of MSCs derived from a heathy environment can be in the near future a smart approach to treat bone loss in the context of regenerative dentistry for oral rehabilitation of hypertensive patients., (© 2024 Wiley Periodicals LLC.)
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- 2024
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8. Bone Regeneration and Repair Materials.
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Beloti MM and Rosa AL
- Abstract
Bone tissue has a remarkable ability to regenerate following injury and trauma [...].
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- 2024
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9. Mesenchymal stem cell-based therapy for osteoporotic bones: Effects of the interaction between cells from healthy and osteoporotic rats on osteoblast differentiation and bone repair.
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Souza ATP, Freitas GP, Lopes HB, Weffort D, Adolpho LF, Gomes MPO, Oliveira FS, Almeida ALG, Beloti MM, and Rosa AL
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- Humans, Rats, Animals, Osteogenesis, Bone and Bones metabolism, Cell Differentiation genetics, Osteoblasts metabolism, Cells, Cultured, Mesenchymal Stem Cells, Osteoporosis metabolism
- Abstract
Aims: Cell therapy utilizing mesenchymal stem cells (MSCs) from healthy donors (HE-MSCs) is a promising strategy for treating osteoporotic bone defects. This study investigated the effects of interaction between HE-MSCs and MSCs from osteoporotic donors (ORX-MSCs) on osteoblast differentiation of MSCs and of HE-MSCs on bone formation in calvarial defects of osteoporotic rats., Materials and Methods: Osteoporosis was induced by orchiectomy (ORX) and its effects on the bone were evaluated by femur microtomography (μCT) and osteoblast differentiation of bone marrow MSCs. HE- and ORX-MSCs were cocultured, and osteoblast differentiation was evaluated using genotypic and phenotypic parameters. HE-MSCs were injected into the calvarial defects of osteoporotic rats, and bone formation was evaluated by μCT, histology, and gene expression of osteoblast markers., Key Findings: ORX-induced osteoporosis was revealed by reduced bone morphometric parameters and osteoblast differentiation in ORX-MSCs. HE-MSCs partially recovered the osteogenic potential of ORX-MSCs, whereas HE-MSCs were mildly affected by ORX-MSCs. Additionally, the bone morphogenetic protein and wingless-related integration site signaling pathway components were similarly modulated in cocultures involving ORX-MSCs. HE-MSCs induced meaningful bone formation, highlighting the effectiveness of cell therapy even in osteoporotic bones., Significance: These results provide new perspectives on the development of cell-based therapies to regenerate bone defects in patients with disorders that affect bone tissue., Competing Interests: Declaration of competing interest Alann T. P. Souza, Gileade P. Freitas, Helena B. Lopes, Denise Weffort, Leticia F. Adolpho, Maria P. O. Gomes, Fabiola S. Oliveira, Adriana L. G. Almeida, Marcio M Beloti, Adalberto L. Rosa declare that they have no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)
- Published
- 2024
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10. Effect of the secretome of mesenchymal stem cells overexpressing BMP-9 on osteoblast differentiation and bone repair.
- Author
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Calixto RD, Freitas GP, Souza PG, Ramos JIR, Santos IC, de Oliveira FS, Almeida ALG, Rosa AL, and Beloti MM
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- Animals, Mice, Bone Morphogenetic Protein 2 metabolism, Cell Differentiation, Cells, Cultured, Growth Differentiation Factor 2 genetics, Osteoblasts metabolism, Secretome, Mesenchymal Stem Cells metabolism, Osteogenesis
- Abstract
The secretome present in the conditioned medium (CM) of mesenchymal stem cells (MSCs) is a promising tool to be used in therapies to promote bone regeneration. Considering the high osteogenic potential of the bone morphogenetic protein 9 (BMP-9), we hypothesized that the secretome of MSCs overexpressing BMP-9 (MSCs
BMP-9 ) enhances the osteoblast differentiation of MSCs and the bone formation in calvarial defects. CM of either MSCsBMP-9 (CM-MSCsBMP-9 ) or MSCs without BMP-9 overexpression (CM-MSCsVPR ) were obtained at different periods. As the CM-MSCsBMP-9 generated after 1 h presented the highest BMP-9 concentration, CM-MSCsBMP-9 and CM-MSCsVPR were collected at this time point and used to culture MSCs and to be injected into mouse calvarial defects. The CM-MSCsBMP-9 enhanced the osteoblast differentiation of MSC by upregulating RUNX2, alkaline phosphatase (ALP) and osteopontin protein expression, and ALP activity, compared with CM-MSCsVPR . The CM-MSCsBMP-9 also enhanced the bone repair of mouse calvarial defects, increasing bone volume, bone volume/total volume, bone surface, and trabecular number compared with untreated defects and defects treated with CM-MSCsVPR or even with MSCsBMP-9 themselves. In conclusion, the potential of the MSCBMP-9 -secretome to induce osteoblast differentiation and bone formation shed lights on novel cell-free-based therapies to promote bone regeneration of challenging defects., (© 2023 Wiley Periodicals LLC.)- Published
- 2023
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11. Disruption of TNF-α signaling improves osteoblastic differentiation of adipose-derived mesenchymal stem cells and bone repair.
- Author
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Bighetti-Trevisan RL, Bueno NP, Souza ATP, Marques MM, Rosa AL, Beloti MM, and Ferraz EP
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- Animals, Mice, Cell Differentiation, Cells, Cultured, Core Binding Factor Alpha 1 Subunit genetics, Core Binding Factor Alpha 1 Subunit metabolism, Mice, Knockout, Osteoblasts, Mesenchymal Stem Cells, Osteogenesis, Tumor Necrosis Factor-alpha genetics, Tumor Necrosis Factor-alpha metabolism
- Abstract
Adipose tissue is an attractive source of mesenchymal stem cells (at-MSCs), but their low osteogenic potential limits their use in bone regeneration. Adipose tissue plays a role in pro-inflammatory diseases by releasing cytokines with a catabolic effect on bone, such as tumor necrosis factor-alpha (TNF-α). Thus, we hypothesized that endogenous TNF-α could have a negative effect on at-MSC differentiation into osteoblasts. Short interfering RNAs (siRNAs) targeting TNF-α receptors (siR1, siR2, and si1R/R2) were transfected into at-MSCs, and cell differentiation was assessed by measuring the expression of bone markers, ALP activity, and mineralized matrix. Scrambled was used as Control. Knockout at-MSCs (KOR1/R2) was injected in mice calvaria defects, and bone formation was evaluated by microtomography and histological analysis. Data were compared by Kruskal-Wallis or analysis of variance (5%). The expression of bone markers confirmed that at-MSCs differentiate less than bone marrow MSCs. In silenced cells, the expression of Alp, Runx2, and Opn was generally higher compared to Control. ALP, RUNX2, and OPN were expressed at elevated levels in silenced groups, most notably at-MSCs-siR1/R2. ALP was detected at high levels in at-MSCs-siR1/R2 and in-MSCs-siR1, followed by an increase in mineralized nodules in at-MSCs-siR1/R2. As the morphometric parameters increased, the groups treated with KOR1/R2 exhibited slight bone formation near the edges of the defects. Endogenous TNF-α inhibits osteoblast differentiation and activity in at-MSCs, and its disruption increases bone formation. While opening a path of investigation, that may lead to the development of new treatments for bone regeneration using at-MSC-based therapies., (© 2023 Wiley Periodicals LLC.)
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- 2023
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12. Effect of osteoblasts on osteoclast differentiation and activity induced by titanium with nanotopography.
- Author
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Bighetti-Trevisan RL, Ferraz EP, Silva MBF, Zatta GC, de Almeida MB, Rosa AL, and Beloti MM
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- Hydrogen Peroxide pharmacology, Osteoblasts, Cell Differentiation, Osteoclasts, Titanium pharmacology, Titanium metabolism
- Abstract
Titanium with nanotopography (Ti Nano) favors osteoblast differentiation and attenuates the osteoclast inhibitory effects on osteoblasts. Because the interactions between nanotopography and osteoclasts are underexplored, the aims of this study were to evaluate the effects of Ti Nano on osteoclast differentiation and activity, and the influence of osteoblasts on osteoclast-Ti Nano interaction. The discs were conditioned with a mixture of 10 N H
2 SO4 and 30% aqueous H2 O2 to create Ti Nano and non-conditioned Ti discs were used as control (Ti Control). Osteoclasts were cultured on Ti Control and Ti Nano in the presence of osteoblasts in an indirect co-culture system. Also, osteoclasts were cultured on polystyrene and calcium phosphate plates in conditioned media by osteoblasts grown on Ti Control and Ti Nano. While Ti Control exhibited an irregular and smooth surface, Ti Nano presented nanopores distributed throughout the whole surface. Additionally, anisotropy was higher on Ti Nano than Ti Control. Nanotopography favored the gene expression of osteoclast markers but inhibited osteoclast differentiation and activity, and the presence of osteoblasts enhanced the effects of Ti Nano on osteoclasts. Such findings were mimicked by conditioned medium of osteoblasts cultured on Ti Nano, which reduced the osteoclast differentiation and activity. In conclusion, our results indicated that nanotopography regulates osteoblast-osteoclast crosstalk and further investigations should focus the impact of these bone cell interactions on Ti osseointegration., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)- Published
- 2023
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13. Effect of Mesenchymal Stem Cells Overexpressing BMP-9 Primed with Hypoxia on BMP Targets, Osteoblast Differentiation and Bone Repair.
- Author
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Paz JERM, Adolpho LF, Ramos JIR, Bighetti-Trevisan RL, Calixto RD, Oliveira FS, Almeida ALG, Beloti MM, and Rosa AL
- Abstract
Bone formation is driven by many signaling molecules including bone morphogenetic protein 9 (BMP-9) and hypoxia-inducible factor 1-alpha (HIF-1α). We demonstrated that cell therapy using mesenchymal stem cells (MSCs) overexpressing BMP-9 (MSCs
+BMP-9 ) enhances bone formation in calvarial defects. Here, the effect of hypoxia on BMP components and targets of MSCs+BMP-9 and of these hypoxia-primed cells on osteoblast differentiation and bone repair was evaluated. Hypoxia was induced with cobalt chloride (CoCl2 ) in MSCs+BMP-9 , and the expression of BMP components and targets was evaluated. The paracrine effects of hypoxia-primed MSCs+BMP-9 on cell viability and migration and osteoblast differentiation were evaluated using conditioned medium. The bone formation induced by hypoxia-primed MSCs+BMP-9 directly injected into rat calvarial defects was also evaluated. The results demonstrated that hypoxia regulated BMP components and targets without affecting BMP-9 amount and that the conditioned medium generated under hypoxia favored cell migration and osteoblast differentiation. Hypoxia-primed MSCs+BMP-9 did not increase bone repair compared with control MSCs+BMP-9 . Thus, despite the lack of effect of hypoxia on bone formation, the enhancement of cell migration and osteoblast differentiation opens windows for further investigations on approaches to modulate the BMP-9-HIF-1α circuit in the context of cell-based therapies to induce bone regeneration.- Published
- 2023
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14. Normoglycemia partially recovers the disrupted osteoblast differentiation of mesenchymal stem cells induced by type 1 but not type 2 diabetes mellitus.
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Weffort D, Adolpho LF, Souza ATP, Freitas GP, Lopes HB, Oliveira FS, Bighetti-Trevisan RL, Pitol-Palin L, Matsushita DH, Okamoto R, Beloti MM, and Rosa AL
- Subjects
- Rats, Animals, Cells, Cultured, Osteogenesis genetics, Cell Differentiation, Osteoblasts metabolism, Diabetes Mellitus, Type 1 metabolism, Diabetes Mellitus, Type 2 metabolism, Mesenchymal Stem Cells metabolism
- Abstract
Type 1 (T1DM) and type 2 (T2DM) diabetes mellitus are characterized by changes in glucose metabolism and cause bone damage via a variety of mechanisms, including effects on osteoblasts. We aimed to evaluate the osteoblast differentiation of mesenchymal stem cells (MSCs) from rats with T1DM or T2DM and the effects of removing the hyperglycemic stimulus on the osteogenic potential of these cells. MSCs from healthy rats were cultured in normoglycemic medium, whereas MSCs from rats with T1DM or T2DM were cultured in hyperglycemic or normoglycemic medium. T1DM and T2DM reduced osteoblast differentiation of MSCs grown in hyperglycemic media, with T1DM having a more pronounced effect, as evidenced by alkaline phosphatase activity, RUNX2 protein expression, and extracellular matrix mineralization, and modulated the gene expression of several components of the bone morphogenetic protein signaling pathway. The restoration of the normoglycemic environment partially recovers the osteogenic potential of MSCs from rats with T1DM but not with T2DM. Our findings highlight the need for specific therapies to treat T1DM- or T2DM-induced bone loss, as both disrupt osteoblast differentiation at distinct levels and likely through different mechanisms., (© 2023 Wiley Periodicals LLC.)
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- 2023
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15. Mesenchymal Stem Cells Combined with a P(VDF-TrFE)/BaTiO 3 Scaffold and Photobiomodulation Therapy Enhance Bone Repair in Rat Calvarial Defects.
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Adolpho LF, Ribeiro LMS, Freitas GP, Lopes HB, Gomes MPO, Ferraz EP, Gimenes R, Beloti MM, and Rosa AL
- Abstract
Background: Tissue engineering and cell therapy have been the focus of investigations on how to treat challenging bone defects. This study aimed to produce and characterize a P(VDF-TrFE)/BaTiO
3 scaffold and evaluate the effect of mesenchymal stem cells (MSCs) combined with this scaffold and photobiomodulation (PBM) on bone repair., Methods and Results: P(VDF-TrFE)/BaTiO3 was synthesized using an electrospinning technique and presented physical and chemical properties suitable for bone tissue engineering. This scaffold was implanted in rat calvarial defects (unilateral, 5 mm in diameter) and, 2 weeks post-implantation, MSCs were locally injected into these defects ( n = 12/group). Photobiomodulation was then applied immediately, and again 48 and 96 h post-injection. The μCT and histological analyses showed an increment in bone formation, which exhibited a positive correlation with the treatments combined with the scaffold, with MSCs and PBM inducing more bone repair, followed by the scaffold combined with PBM, the scaffold combined with MSCs, and finally the scaffold alone (ANOVA, p ≤ 0.05)., Conclusions: The P(VDF-TrFE)/BaTiO3 scaffold acted synergistically with MSCs and PBM to induce bone repair in rat calvarial defects. These findings emphasize the need to combine a range of techniques to regenerate large bone defects and provide avenues for further investigations on innovative tissue engineering approaches.- Published
- 2023
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16. Association of mesenchymal stem cells derived from bone marrow and adipose tissue enhances bone repair in rat calvarial defects.
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Campos Totoli GG, Bighetti-Trevisan RL, Freitas GP, Adolpho LF, Golçalves Almeida AL, Loyola Barbosa AC, Reis Ramos JI, Beloti MM, and Rosa AL
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- Rats, Animals, Adipose Tissue, Osteogenesis, Bone Regeneration, Cell Differentiation, Bone Marrow Cells, Cells, Cultured, Bone Marrow, Mesenchymal Stem Cells
- Abstract
Aim: We evaluated the bone repair induced by MSCs from adipose tissue (AT-MSCs) and bone marrow (BM-MSCs) injected into rat calvarial defects at two time points. Methods & results: Both cell populations expressed MSC surface markers and differentiated into adipocytes and osteoblasts. μCT showed that the combination of cells from distinct sources exhibited synergistic effects to increase bone repair with an advantage when BM-MSCs were injected prior to AT-MSCs. The higher osteogenic potential of these MSC combinations was demonstrated using an in vitro coculture system where BM-MSCs and AT-MSCs association induced higher ALP activity in MC3T3-E1 cells. Conclusion: Our findings may drive new approaches to treat bone defects and shed light on the complexity of the mechanisms involved in bone regeneration.
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- 2023
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17. Collagen/κ-Carrageenan-Based Scaffolds as Biomimetic Constructs for In Vitro Bone Mineralization Studies.
- Author
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Nogueira LFB, Cruz MAE, de Melo MT, Maniglia BC, Caroleo F, Paolesse R, Lopes HB, Beloti MM, Ciancaglini P, Ramos AP, and Bottini M
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- Carrageenan, Collagen chemistry, Tissue Engineering methods, Osteoblasts, Tissue Scaffolds chemistry, Calcification, Physiologic, Biomimetics
- Abstract
Tissue engineering offers attractive strategies to develop three-dimensional scaffolds mimicking the complex hierarchical structure of the native bone. The bone is formed by cells incorporated in a molecularly organized extracellular matrix made of an inorganic phase, called biological apatite, and an organic phase mainly made of collagen and noncollagenous macromolecules. Although many strategies have been developed to replicate the complexity of bone at the nanoscale in vitro , a critical challenge has been to control the orchestrated process of mineralization promoted by bone cells in vivo and replicate the anatomical and biological properties of native bone. In this study, we used type I collagen to fabricate mineralized scaffolds mimicking the microenvironment of the native bone. The sulfated polysaccharide κ-carrageenan was added to the scaffolds to fulfill the role of noncollagenous macromolecules in the organization and mineralization of the bone matrix and cell adhesion. Scanning electron microscopy images of the surface of the collagen/κ-carrageenan scaffolds showed the presence of a dense and uniform network of intertwined fibrils, while images of the scaffolds' lateral sides showed the presence of collagen fibrils with a parallel alignment, which is characteristic of dense connective tissues. MC3T3-E1 osteoblasts were cultured in the collagen scaffolds and were viable after up to 7 days of culture, both in the absence and in the presence of κ-carrageenan. The presence of κ-carrageenan in the collagen scaffolds stimulated the maturation of the cells to a mineralizing phenotype, as suggested by the increased expression of key genes related to bone mineralization, including alkaline phosphatase ( Alp ), bone sialoprotein ( Bsp ), osteocalcin ( Oc ), and osteopontin ( Opn ), as well as the ability to mineralize the extracellular matrix after 14 and 21 days of culture. Taken together, the results described in this study shed light on the potential use of collagen/κ-carrageenan scaffolds to study the role of the structural organization of bone-mimetic synthetic matrices in cell function.
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- 2023
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18. Effects of Modulation of the Hedgehog and Notch Signaling Pathways on Osteoblast Differentiation Induced by Titanium with Nanotopography.
- Author
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Souza PG, Adolpho LF, Lopes HB, Weffort D, Souza ATP, Oliveira FS, Rosa AL, and Beloti MM
- Abstract
Background: The events of bone formation and osteoblast/titanium (Ti) interactions may be affected by Hedgehog and Notch signalling pathways. Herein, we investigated the effects of modulation of these signalling pathways on osteoblast differentiation caused by the nanostructured Ti (Ti-Nano) generated by H
2 SO4 /H2 O2 ., Methods: Osteoblasts from newborn rat calvariae were cultured on Ti-Control and Ti-Nano in the presence of the Hedgehog agonist purmorphamine or antagonist cyclopamine and of the Notch antagonist N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) or agonist bexarotene. Osteoblast differentiation was evaluated by alkaline phosphatase activity and mineralization, and the expression of Hedgehog and Notch receptors was also evaluated., Results: In general, purmorphamine and DAPT increased while cyclopamine and bexarotene decreased osteoblast differentiation and regulated the receptor expression on both Ti surfaces, with more prominent effects on Ti-Nano. The purmorphamine and DAPT combination exhibited synergistic effects on osteoblast differentiation that was more intense on Ti-Nano., Conclusion: Our results indicated that the Hedgehog and Notch signalling pathways drive osteoblast/Ti interactions more intensely on nanotopography. We also demonstrated that combining Hedgehog activation with Notch inhibition exhibits synergistic effects on osteoblast differentiation, especially on Ti-Nano. The uncovering of these cellular mechanisms contributes to create strategies to control the process of osseointegration based on the development of nanostructured surfaces.- Published
- 2023
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19. Curcumin-loaded carrageenan nanoparticles: Fabrication, characterization, and assessment of the effects on osteoblasts mineralization.
- Author
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Nogueira LFB, Cruz MAE, Tovani CB, Lopes HB, Beloti MM, Ciancaglini P, Bottini M, and Ramos AP
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- Carrageenan chemistry, Drug Carriers chemistry, Drug Liberation, Osteoblasts, Particle Size, Curcumin chemistry, Nanoparticles chemistry
- Abstract
The use of Curcumin (CR) as a bioactive molecule to prevent and treat inflammation- related diseases is widespread. However, the high hydrophobicity hinders the in vivo bioavailability of CR, reducing its therapeutic index. In the present study, we described the use of nanoparticles (NPs) made of kappa-carrageenan (κ-Carr), a sulphated polysaccharide, as cost-effective, biodegradable and biocompatible CR carriers. CR-loaded κ-Carr nanoparticles (CR@Carr NPs) were prepared by mixing a κ-Carr aqueous solution with a CR ethanolic solution. The final suspension was centrifuged and re-suspended in phosphate buffer solution. The NPs' size was tuned by changing the concentration of the polysaccharide. CR@CarrNPs displayed high CR incorporation efficiency (~80 wt%) and a double-exponential curve of CR release at physiological conditions (37 °C and pH 7.4) with a cumulative drug release of 32 wt% after 24 h for the smaller NP. Our results also showed that CR@CarrNPs were not cytotoxic to osteoblasts at concentrations up to 1 μM. Confocal microscopy images revealed the internalization of CR by the cells guided by the NPs. Cells treated with CR@CarrNPs exhibited higher activity of alkaline phosphatase and higher expression of the main osteogenic genes (Sp7, Col1 and Runx2), and mineralized the extracellular matrix in a higher extent compared to the cells cultivated in absence of the NPs. We posited that these effects were related to the NP-driven internalization of CR by osteoblasts. Our study sheds light on the possible use of CR@CarrNPs as efficient and safe therapeutic tools for the treatment of bone-related diseases., (Copyright © 2022 Elsevier B.V. All rights reserved.)
- Published
- 2022
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20. Human periodontal ligament stem cells with distinct osteogenic potential induce bone formation in rat calvaria defects.
- Author
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Adolpho LF, Lopes HB, Freitas GP, Weffort D, Campos Totoli GG, Loyola Barbosa AC, Freire Assis RI, Silverio Ruiz KG, Andia DC, Rosa AL, and Beloti MM
- Subjects
- Animals, Cell Differentiation, Cell Proliferation, Cells, Cultured, Humans, Rats, Skull, Stem Cells, Osteogenesis, Periodontal Ligament
- Abstract
Aim: This study aimed to evaluate the ability of human periodontal ligament stem cells (PDLSCs) with high (HP-PDLSCs) and low (LP-PDLSCs) osteogenic potential, in addition to mixed cells, to repair bone tissue. Methods: Cell phenotype, proliferation and differentiation were evaluated. Undifferentiated PDLSCs were injected into rat calvarial defects and the new bone was evaluated by μCT, histology and real-time PCR. Results: PDLSCs exhibited a typical mesenchymal stem cell phenotype and HP-PDLSCs showed lower proliferative and higher osteogenic potential than LP-PDLSCs. PDLSCs induced similar bone formation and histological analysis suggests a remodeling process, confirmed by osteogenic and osteoclastogenic markers, especially in tissues derived from defects treated with HP-PDLSCs. Conclusion: PDLSCs induced similar bone formation irrespective of their in vitro osteogenic potential.
- Published
- 2022
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21. Bioactive glass-ceramic for bone tissue engineering: an in vitro and in vivo study focusing on osteoclasts.
- Author
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Bighetti-Trevisan RL, Souza ATP, Tosin IW, Bueno NP, Crovace MC, Beloti MM, Rosa AL, and Ferraz EP
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- Animals, Bone and Bones, Ceramics chemistry, Osteoblasts, Rats, Osteoclasts, Tissue Engineering
- Abstract
Despite the crucial role of osteoclasts in the physiological process of bone repair, most bone tissue engineering strategies have focused on osteoblast-biomaterial interactions. Although Biosilicate® with two crystalline phases (BioS-2P) exhibits osteogenic properties and significant bone formation, its effects on osteoclasts are unknown. This study aimed to investigate the in vitro and in vivo effects of BioS-2P on osteoclast differentiation and activity. RAW 264.7 cells were cultured in osteoclastogenic medium (OCM) or OCM conditioned with BioS-2P (OCM-BioS-2P), and the cell morphology, viability, and osteoclast differentiation were evaluated. BioS-2P scaffolds were implanted into rat calvarial defects, and the bone tissue was evaluated using tartrate-resistant acid phosphatase (TRAP) staining and RT-polymerase chain reaction (PCR) after 2 and 4 weeks to determine the gene expressions of osteoclast markers and compare them with those of the bone grown in empty defects (Control). OCM-BioS-2P favored osteoclast viability and activity, as evidenced by an increase in the TRAP-positive cells and matrix resorption. The bone tissue grown on BioS-2P scaffolds exhibited higher expression of the osteoclast marker genes (Ctsk, Mmp 9, Rank) after 2 and 4 weeks and the RankL/Opg ratio after 2 weeks. Trap gene expression was lower at 2 weeks, and a higher number of TRAP-stained areas were observed in the newly formed bone on BioS-2P scaffolds at both 2 and 4 weeks compared to the Controls. These results enhanced our understanding of the role of bioactive glass-ceramics in bone repair, and highlighted their role in the modulation of osteoclastic activities and promotion of interactions between bone tissues and biomaterials.
- Published
- 2022
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22. Titanium with nanotopography attenuates the osteoclast-induced disruption of osteoblast differentiation by regulating histone methylation.
- Author
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Bighetti-Trevisan RL, Almeida LO, Castro-Raucci LMS, Gordon JAR, Tye CE, Stein GS, Lian JB, Stein JL, Rosa AL, and Beloti MM
- Subjects
- Histones metabolism, Methylation, Osteoblasts, Surface Properties, Osteoclasts metabolism, Titanium pharmacology
- Abstract
The bone remodeling process is crucial for titanium (Ti) osseointegration and involves the crosstalk between osteoclasts and osteoblasts. Considering the high osteogenic potential of Ti with nanotopography (Ti Nano) and that osteoclasts inhibit osteoblast differentiation, we hypothesized that nanotopography attenuate the osteoclast-induced disruption of osteoblast differentiation. Osteoblasts were co-cultured with osteoclasts on Ti Nano and Ti Control and non-co-cultured osteoblasts were used as control. Gene expression analysis using RNAseq showed that osteoclasts downregulated the expression of osteoblast marker genes and upregulated genes related to histone modification and chromatin organization in osteoblasts grown on both Ti surfaces. Osteoclasts also inhibited the mRNA and protein expression of osteoblast markers, and such effect was attenuated by Ti Nano. Also, osteoclasts increased the protein expression of H3K9me2, H3K27me3 and EZH2 in osteoblasts grown on both Ti surfaces. ChIP assay revealed that osteoclasts increased accumulation of H3K27me3 that represses the promoter regions of Runx2 and Alpl in osteoblasts grown on Ti Control, which was reduced by Ti Nano. In conclusion, these data show that despite osteoclast inhibition of osteoblasts grown on both Ti Control and Ti Nano, the nanotopography attenuates the osteoclast-induced disruption of osteoblast differentiation by preventing the increase of H3K27me3 accumulation that represses the promoter regions of some key osteoblast marker genes. These findings highlight the epigenetic mechanisms triggered by nanotopography to protect osteoblasts from the deleterious effects of osteoclasts, which modulate the process of bone remodeling and may benefit the osseointegration of Ti implants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)
- Published
- 2022
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23. Texturized P(VDF-TrFE)/BT membrane enhances bone neoformation in calvaria defects regardless of the association with photobiomodulation therapy in ovariectomized rats.
- Author
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Rufato FCT, de Sousa LG, Scalize PH, Gimenes R, Regalo IH, Rosa AL, Beloti MM, de Oliveira FS, Bombonato-Prado KF, Regalo SCH, and Siéssere S
- Subjects
- Animals, Female, Osteogenesis, Rats, Rats, Wistar, Skull surgery, Titanium, Low-Level Light Therapy
- Abstract
Objectives: The purpose of this investigation was to evaluate in vivo the response of bone tissue to photobiomodulation when associated with texturized P(VDF-TrFE)/BT in calvaria defects of ovariectomized rats., Materials and Methods: Wistar Hannover rats were submitted to ovariectomy/control surgery. Calvaria bone defects of 5-mm diameter were performed after 90 days of ovariectomy. The animals were divided into OVX (without laser (L) and membrane), OVX + P(VDF-TrFE)/BT, OVX + P(VDF-TrFE)/BT + L, and OVX + PTFE + L. It was utilized a low-intensity gallium-aluminum-arsenide laser (GaAlAs) with 780-nm wavelength and 30-J/cm
2 energy density in 12 sessions (120 s). Thirty days after the bone defect the animals were euthanized for histological, microtomographic, and molecular evaluation. Quantitative analysis was analyzed by statistical software for p < 0.05., Results: Histological parameters showed bone tissue formation at the borders of all group defects. The association of photobiomodulation and texturized P(VDF-TrFE)/BT was not synergistic and did not show significant changes in morphometric analysis and biomarkers gene expression. Nevertheless, texturized P(VDF-TrFE)/BT membrane enhanced bone repair regardless of the association with photobiomodulation therapy, with an increase of connectivity density when compared to the OVX + PTFE + L group. The association of photobiomodulation therapy and PTFE was synergistic, increasing the expression of Runx2, Alp, Bsp, Bglap, Sp7, and Rankl, even though not enough to reflect significance in the morphometric parameters., Conclusions: The utilization of texturized P (VDF-TrFE)/BT, regardless of the association with photobiomodulation therapy, enhanced bone repair in an experimental model of osteoporosis., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)- Published
- 2022
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24. Mesenchymal stem cells overexpressing BMP-9 by CRISPR-Cas9 present high in vitro osteogenic potential and enhance in vivo bone formation.
- Author
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Freitas GP, Lopes HB, Souza ATP, Gomes MPO, Quiles GK, Gordon J, Tye C, Stein JL, Stein GS, Lian JB, Beloti MM, and Rosa AL
- Subjects
- Animals, CRISPR-Cas Systems, Cell Differentiation, Cells, Cultured, Rats, Growth Differentiation Factor 2 genetics, Mesenchymal Stem Cells cytology, Osteogenesis genetics
- Abstract
Cell therapy is a valuable strategy for the replacement of bone grafts and repair bone defects, and mesenchymal stem cells (MSCs) are the most frequently used cells. This study was designed to genetically edit MSCs to overexpress bone morphogenetic protein 9 (BMP-9) using Clustered Regularly Interspaced Short Palindromic Repeats/associated nuclease Cas9 (CRISPR-Cas9) technique to generate iMSCs-VPR
BMP-9+ , followed by in vitro evaluation of osteogenic potential and in vivo enhancement of bone formation in rat calvaria defects. Overexpression of BMP-9 was confirmed by its gene expression and protein expression, as well as its targets Hey-1, Bmpr1a, and Bmpr1b, Dlx-5, and Runx2 and protein expression of SMAD1/5/8 and pSMAD1/5/8. iMSCs-VPRBMP-9+ displayed significant changes in the expression of a panel of genes involved in TGF-β/BMP signaling pathway. As expected, overexpression of BMP-9 increased the osteogenic potential of MSCs indicated by increased gene expression of osteoblastic markers Runx2, Sp7, Alp, and Oc, higher ALP activity, and matrix mineralization. Rat calvarial bone defects treated with injection of iMSCs-VPRBMP-9+ exhibited increased bone formation and bone mineral density when compared with iMSCs-VPR- and phosphate buffered saline (PBS)-injected defects. This is the first study to confirm that CRISPR-edited MSCs overexpressing BMP-9 effectively enhance bone formation, providing novel options for exploring the capability of genetically edited cells to repair bone defects., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited part of Springer Nature.)- Published
- 2021
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25. The extracellular matrix protein Agrin is expressed by osteoblasts and contributes to their differentiation.
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Souza ATP, Lopes HB, Oliveira FS, Weffort D, Freitas GP, Adolpho LF, Fernandes RR, Rosa AL, and Beloti MM
- Subjects
- 3T3 Cells, Agrin genetics, Animals, Cell Differentiation, Cells, Cultured, Gene Expression Regulation, Humans, Mice, Mice, Inbred C57BL, Osteoblasts metabolism, Osteogenesis, Agrin analysis, Osteoblasts cytology
- Abstract
The extracellular matrix protein Agrin has been detected in chondrocytes and endosteal osteoblasts but its function in osteoblast differentiation has not been investigated yet. Thus, it is possible that Agrin contributes to osteoblast differentiation and, due to Agrin and wingless-related integration site (Wnt) sharing the same receptor, transmembrane low-density lipoprotein receptor-related protein 4 (Lrp4), and the crosstalk between Wnt and bone morphogenetic protein (BMP) signalling, both pathways could be involved in this Agrin-mediated osteoblast differentiation. Confirming this, Agrin and its receptors Lrp4 and α-dystroglycan (Dag1) were expressed during differentiation of osteoblasts from three different sources. Moreover, the disruption of Agrin impaired the expression of its receptors and osteoblast differentiation, and the treatment with recombinant Agrin slightly increase this process. In addition, whilst Agrin knockdown downregulated the expression of genes related to Wnt and BMP signalling pathways, the addition of Agrin had no effect on these genes. Altogether, these data uncover the contribution of Agrin to osteoblast differentiation and suggest that, at least in part, an Agrin-Wnt-BMP circuit is involved in this process. This makes Agrin a candidate as target for developing new therapeutic strategies to treat bone-related diseases and injuries., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)
- Published
- 2021
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26. Osteoporosis and osteoblasts cocultured with adipocytes inhibit osteoblast differentiation by downregulating histone acetylation.
- Author
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Abuna RPF, Almeida LO, Souza ATP, Fernandes RR, Sverzut TFV, Rosa AL, and Beloti MM
- Subjects
- Acetylation drug effects, Adipocytes drug effects, Adipogenesis drug effects, Animals, Coculture Techniques, Culture Media, Conditioned pharmacology, Male, Models, Biological, Osteoblasts drug effects, Osteogenesis drug effects, Osteoporosis metabolism, Rats, Wistar, Rats, Adipocytes metabolism, Cell Differentiation drug effects, Down-Regulation drug effects, Histones metabolism, Osteoblasts metabolism, Osteoporosis pathology
- Abstract
Osteoporosis is characterized by decreased bone mass and adipocyte accumulation within the bone marrow that inhibits osteoblast maturation, leading to a high risk of fractures. Thus, we hypothesized that osteoblasts, besides being negatively affected by interacting with adipocytes, reduce the differentiation of neighboring osteoblasts through the same mechanisms that affect osteoblasts under osteoporotic conditions. We investigated the effect of osteoporosis on osteoblast differentiation and the effect of the conditioned medium of osteoblasts cocultured with adipocytes on the differentiation of other osteoblasts. Osteoporosis was induced by orchiectomy in rats and bone marrow mesenchymal stromal cells (MSCs) were differentiated into osteoblasts. Also, the bone marrow and adipose tissue MSCs were obtained from healthy rats and differentiated into osteoblasts and adipocytes, respectively. Messenger RNA expression, in situ alkaline phosphatase activity, and mineralization confirmed the inhibitory effect of osteoporosis on osteoblast differentiation. This harmful effect was mimicked by the in vitro model using the conditioned medium and it was demonstrated that osteoblasts keep the memory of the negative impact of interacting with adipocytes, revealing an unknown mechanism relevant to the osteoporotic bone loss. Finally, we showed the involvement of acetyl-histone 3 (AcH3) in bone homeostasis as its reduction induced by osteoporosis and conditioned medium impaired osteoblast differentiation. The AcH3 involvement was proved by treating osteoblasts with Trichostatin A that recovered the AcH3 expression and osteoblast differentiation capacity in both situations. Together, our findings indicated that AcH3 might be a target for future studies focused on epigenetic-based therapies to treat bone diseases., (© 2020 Wiley Periodicals LLC.)
- Published
- 2021
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27. Jabuticaba peel extract modulates adipocyte and osteoblast differentiation of MSCs from healthy and osteoporotic rats.
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Souza ATP, Freitas GP, Lopes HB, Totoli GGC, Tarone AG, Marostica-Junior MR, Rosa AL, and Beloti MM
- Subjects
- Adipocytes drug effects, Adipocytes metabolism, Adipogenesis drug effects, Adipogenesis physiology, Animals, Bone Marrow drug effects, Bone Marrow metabolism, Bone Marrow Cells drug effects, Bone Marrow Cells metabolism, Bone and Bones drug effects, Bone and Bones pathology, Cell Differentiation drug effects, Cells, Cultured, Female, Mesenchymal Stem Cells drug effects, Osteoblasts drug effects, Osteoblasts metabolism, Osteogenesis drug effects, Osteoporosis metabolism, Ovariectomy, Rats, Wistar, Rats, Adipocytes cytology, Mesenchymal Stem Cells cytology, Osteoblasts cytology, Osteoporosis pathology, Plant Extracts pharmacology
- Abstract
Introduction: The jabuticaba peel extract (JPE) contains bioactive compounds that regulate fat metabolism. Because the negative correlation between fat accumulation and bone formation in bone marrow, we hypothesized that JPE inhibits adipocyte as well as favors osteoblast differentiation of mesenchymal stromal cells (MSCs) under healthy and osteoporotic conditions, a disease that display an imbalance between adipocyte and osteoblast differentiation resulting in reduced bone mass., Material and Methods: To test these hypotheses, bone marrow MSCs were harvested from healthy and osteoporotic rats and cultured in adipogenic and osteogenic media with three concentrations of JPE, 0.25, 5 and 10 µg/ml, and vehicle (control). After selecting the most efficient concentrations of JPE, we used them to evaluate adipocyte and osteoblast differentiation of MSCs from both sources., Results: We observed that, in general, JPE inhibited adipocyte differentiation of MSCs with more pronounced effects in cells from healthy than osteoporotic rats. In addition, JPE increased osteoblast differentiation, exhibiting a slightly higher osteogenic potential on MSCs from osteoporotic compared to healthy condition., Conclusion: Our results demonstrated that JPE drives MSCs to inhibit adipocyte differentiation and toward osteoblast differentiation under healthy and osteoporotic conditions. These findings pave the way for further translational studies to investigate the therapeutic possibilities of JPE in both prevention and treatment of osteoporosis.
- Published
- 2021
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28. Green tea extract rich in epigallocatechin gallate impairs alveolar bone loss in ovariectomized rats with experimental periodontal disease.
- Author
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Vargas-Sanchez PK, Pitol DL, de Sousa LG, Beloti MM, Rosa AL, Rossi AC, Siéssere S, and Bombonato-Prado KF
- Subjects
- Animals, Bone Density drug effects, Catechin analogs & derivatives, Female, Osteoporosis pathology, Periodontal Diseases pathology, Rats, Rats, Wistar, Antioxidants pharmacology, Periodontal Diseases drug therapy, Plant Extracts pharmacology
- Abstract
Periodontal disease and osteoporosis are characterized by bone resorption, and researchers have shown an association between these two diseases through increasing loss of systemic bone mass and triggering alveolar bone loss. Green tea is a common and easily accessible beverage, and evidences show that flavonoid epigallocatechin gallate (EGCG) could decrease bone loss in pathologies such as osteoporosis and periodontal disease. In order to verify its possible effects and apply them in the treatment and prevention of these diseases, this investigation aimed to evaluate the influence of green tea extract (GTE) on bone metabolism of ovariectomized rats after experimental periodontal disease (EPD) by histological, morphological and microtomographic parameters. Wistar female rats were divided into Sham, Sham + EPD, Sham + EPD + GTE, OVX, OVX + EPD and OVX + EPD + GTE groups. Immediately after surgery, gavage administration of 50 mg/kg of green tea extract (GTE) was performed for 60 days, with subsequent induction of periodontal disease by ligature 15 days before euthanasia. Mandible and femur samples were collected for histological, morphometric and microtomographic analysis. The results were analysed by means of statistical software with significance set at 5%. Histological and morphometric analysis showed a significant decrease in alveolar and femoral trabecular bone loss in groups that received GTE. Microtomographic results showed that trabecular thickness and bone surface density values in alveolar bone interradicular septum of the OVX + EPD + GTE groups were similar to the Sham group. The results obtained suggest that green tea extract may improve bone metabolism in osteoporotic rats with periodontal disease., (© 2020 Company of the International Journal of Experimental Pathology (CIJEP).)
- Published
- 2020
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29. Frizzled 6 disruption suppresses osteoblast differentiation induced by nanotopography through the canonical Wnt signaling pathway.
- Author
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Abuna RPF, Oliveira FS, Adolpho LF, Fernandes RR, Rosa AL, and Beloti MM
- Subjects
- Animals, Cell Line, Metal Nanoparticles, Mice, Osteogenesis physiology, Surface Properties, Cell Differentiation physiology, Frizzled Receptors metabolism, Osteoblasts metabolism, Titanium chemistry, Titanium pharmacology, Wnt Signaling Pathway physiology
- Abstract
This study aimed to investigate if wingless-related integration site (Wnt) signaling participates in the high osteogenic potential of titanium with nanotopography (Ti-Nano). We showed that among the several components of the Wnt signaling pathway, Frizzled 6 (Fzd6) was the transcript most intensely modulated by nanotopography compared with the untreated Ti surface (Ti-Machined). Then, we investigated whether and how Fzd6 participates in the regulation of osteoblast differentiation caused by nanotopography. The Fzd6 silencing with CRISPR-Cas9 transfection in MC3T3-E1 cells induced a more pronounced inhibition of osteoblast differentiation of cells cultured on nanotopography than those cultured on Ti-Machined. The analysis of the expression of calcium-calmodulin-dependent protein kinase II and β-catenin demonstrated that Fzd6 disruption inhibited the osteoblast differentiation induced by Ti-Nano by preventing the activation of Wnt/β-catenin but not that of Wnt/Ca
2+ signaling, which is usually triggered by the receptor Fzd6. These findings elucidate the biological function of Fzd6 as a receptor that triggers Wnt/β-catenin signaling and the cellular mechanisms modulated by nanotopography during osteoblast differentiation., (© 2020 Wiley Periodicals, Inc.)- Published
- 2020
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30. Role of embryonic origin on osteogenic potential and bone repair capacity of rat calvarial osteoblasts.
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Souza ATP, Lopes HB, Freitas GP, Ferraz EP, Oliveira FS, Almeida ALG, Weffort D, Beloti MM, and Rosa AL
- Subjects
- Animals, Animals, Newborn, Biomarkers metabolism, Cell Differentiation, Cell Proliferation genetics, Cells, Cultured, Gene Expression Regulation, Rats, Wistar, X-Ray Microtomography, Osteoblasts cytology, Osteogenesis genetics, Skull embryology, Wound Healing genetics
- Abstract
Introduction: The aim of this study was to evaluate the in vitro osteogenic potential of osteoblasts from neural crest-derived frontal bone (OB-NC) and mesoderm-derived parietal bone (OB-MS) and the bone formation induced by them when injected into calvarial defects., Materials and Methods: Calvarial bones were collected from newborn Wistar rats (3-day old) and characterized as frontal and parietal prior to OB-NC and OB-MS harvesting. The cells were cultured, and several parameters of osteoblast differentiation were evaluated. These cells, or PBS without cells (control), were locally injected into 5-mm rat calvarial defects (5 × 10
6 cells/defect) and after 4 weeks bone formation was evaluated by morphometric and histological analyses., Results: The characterization of frontal and parietal bones assured the different embryonic origin of both cell populations, OB-NC and OB-MS. The OB-NC presented higher proliferation while the OB-MS presented higher alkaline phosphatase (ALP) activity, extracellular matrix mineralization and gene expression of runt-related transcription factor 2, Alp, bone sialoprotein and osteocalcin revealing their high osteogenic potential. µCT analysis indicated that there was higher amount of bone formation in defects injected with both OB-NC and OB-MS compared to the control. Moreover, the bone tissue formed by both cells displayed the same histological characteristics., Conclusions: Despite the distinct in vitro osteogenic potential, OB-NC and OB-MS induced similar bone repair in a rat calvarial defect model. Thus, osteoblasts, irrespective of their in vitro osteogenic potential linked to embryonic origins, seem to be suitable for cell-based therapies aiming to repair bone defects.- Published
- 2020
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31. Mesenchymal Stromal Cells Derived from Bone Marrow and Adipose Tissue: Isolation, Culture, Characterization and Differentiation.
- Author
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Freitas GP, Souza ATP, Lopes HB, Trevisan RLB, Oliveira FS, Fernandes RR, Ferreira FU, Ros FA, Beloti MM, and Rosa AL
- Abstract
Since their discovery, mesenchymal stromal cells (MSCs) have received a lot of attention, mainly due to their self-renewal potential and multilineage differentiation capacity. For these reasons, MSCs are a useful tool in cell biology and regenerative medicine. In this article, we describe protocols to isolate MSCs from bone marrow (BM-MSCs) and adipose tissues (AT-MSCs), and methods to culture, characterize, and differentiate MSCs into osteoblasts, adipocytes, and chondrocytes. After the harvesting of cells from bone marrow by flushing the femoral diaphysis and enzymatic digestion of abdominal and inguinal adipose tissues, MSCs are selected by their adherence to the plastic tissue culture dish. Within 7 days, MSCs reach 70% confluence and are ready to be used in subsequent experiments. The protocols described here are easy to perform, cost-efficient, require minimal time, and yield a cell population rich in MSCs., Competing Interests: Competing interestsThe authors declare no conflict of interest., (Copyright © 2020 The Authors; exclusive licensee Bio-protocol LLC.)
- Published
- 2020
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32. Effect of focal adhesion kinase inhibition on osteoblastic cells grown on titanium with different topographies.
- Author
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Lopes HB, Souza ATP, Freitas GP, Elias CN, Rosa AL, and Beloti MM
- Subjects
- Animals, Cell Differentiation drug effects, Cell Proliferation drug effects, Cells, Cultured, Focal Adhesion Protein-Tyrosine Kinases analysis, Focal Adhesion Protein-Tyrosine Kinases chemistry, Gene Expression, Integrins analysis, Microscopy, Electron, Scanning, Osseointegration drug effects, Osteoblasts physiology, Quinolones chemistry, Rats, Wistar, Real-Time Polymerase Chain Reaction, Signal Transduction, Sulfones chemistry, Surface Properties, Focal Adhesion Protein-Tyrosine Kinases antagonists & inhibitors, Osteoblasts drug effects, Quinolones pharmacology, Sulfones pharmacology, Titanium chemistry
- Abstract
Objective: The present study aimed to investigate the participation of focal adhesion kinases (FAK) in interactions between osteoblastic cells and titanium (Ti) surfaces with three different topographies, namely, untreated (US), microstructured (MS), and nanostructured (NS)., Methodology: Osteoblasts harvested from the calvarial bones of 3-day-old rats were cultured on US, MS and NS discs in the presence of PF-573228 (FAK inhibitor) to evaluate osteoblastic differentiation. After 24 h, we evaluated osteoblast morphology and vinculin expression, and on day 10, the following parameters: gene expression of osteoblastic markers and integrin signaling components, FAK protein expression and alkaline phosphatase (ALP) activity. A smooth surface, porosities at the microscale level, and nanocavities were observed in US, MS, and NS, respectively., Results: FAK inhibition decreased the number of filopodia in cells grown on US and MS compared with that in NS. FAK inhibition decreased the gene expression of Alp, bone sialoprotein, osteocalcin, and ALP activity in cells grown on all evaluated surfaces. FAK inhibition did not affect the gene expression of Fak, integrin alpha 1 ( Itga1 ) and integrin beta 1 ( Itgb1 ) in cells grown on MS, increased the gene expression of Fak in cells grown on NS, and increased the gene expression of Itga1 and Itgb1 in cells grown on US and NS. Moreover, FAK protein expression decreased in cells cultured on US but increased in cells cultured on MS and NS after FAK inhibition; no difference in the expression of vinculin was observed among cells grown on all surfaces., Conclusions: Our data demonstrate the relevance of FAK in the interactions between osteoblastic cells and Ti surfaces regardless of surface topography. Nanotopography positively regulated FAK expression and integrin signaling pathway components during osteoblast differentiation. In this context, the development of Ti surfaces with the ability to upregulate FAK activity could positively impact the process of implant osseointegration.
- Published
- 2020
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33. Inhibitory effects of dabigatran etexilate, a direct thrombin inhibitor, on osteoclasts and osteoblasts.
- Author
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Rocha AL, Bighetti-Trevisan RL, Duffles LF, de Arruda JAA, Taira TM, Assis BRD, Macari S, Diniz IMA, Beloti MM, Rosa AL, Fukada SY, Goulart GAC, Ribeiro DD, Abreu LG, and Silva TA
- Subjects
- Animals, Anticoagulants pharmacology, Mice, Osteoblasts, Osteoclasts, Thrombin, Antithrombins, Dabigatran pharmacology
- Abstract
Introduction: Anticoagulants are widely used in orthopedic surgery to decrease the risk of deep vein thrombosis. While significant bone impairment is induced by long-term heparin therapy, little is known about the effects of direct oral anticoagulants (DOACs). Herein, we investigated the effects of dabigatran etexilate (Pradaxa®), a DOAC inhibitor of thrombin, on bone cells using in vitro and ex vivo cell culture models., Materials and Methods: Osteoblasts and osteoclasts exposed to different concentrations of dabigatran etexilate and untreated cells were assayed for cell differentiation and activity. Favorable osteogenic conditions for osteoblasts were tested using titanium with nanotopography (Ti-Nano). In addition, mice treated with a dabigatran etexilate solution had bone marrow cells analyzed for the ability to generate osteoclasts., Results: Dabigatran etexilate at concentrations of 1 μg/mL and 2 μg/mL did not impact osteoclast or osteoblast viability. The drug inhibited osteoclast differentiation and activity as observed by the reduction of TRAP+ cells, resorption pits and gene and protein expression of cathepsin K. Consistently, osteoclasts from mice treated with dabigatran showed decreased area, resorptive activity, as well as gene and protein expression of cathepsin K. In osteoblast cultures, grown both on polystyrene and Ti-Nano, dabigatran etexilate reduced alkaline phosphatase (ALP) activity, matrix mineralization, gene expression of ALP and osteocalcin., Conclusions: Dabigatran etexilate inhibited osteoclast differentiation in ex vivo and in vitro models in a dose-dependent manner. Moreover, the drug reduced osteoblast activity even under optimal osteogenic conditions. This study provides new evidence regarding the negative overall impact of DOACs on bone cells., (Copyright © 2019 Elsevier Ltd. All rights reserved.)
- Published
- 2020
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34. Effect of cell therapy with osteoblasts differentiated from bone marrow or adipose tissue stromal cells on bone repair.
- Author
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Freitas GP, Lopes HB, P Souza AT, F P Oliveira PG, G Almeida AL, Coelho PG, Ferreira FU, Covas DT, Beloti MM, and Rosa AL
- Subjects
- Animals, Bone and Bones injuries, Cell Differentiation, Male, Osteogenesis, Rats, Rats, Wistar, Tissue Engineering methods, Bone Marrow Cells cytology, Bone Regeneration, Bone and Bones cytology, Cell- and Tissue-Based Therapy methods, Mesenchymal Stem Cell Transplantation methods, Mesenchymal Stem Cells cytology, Osteoblasts cytology
- Abstract
Aim: The aim of this study was to investigate the effect of local injection of osteoblasts differentiated from bone marrow (BM-OB) or adipose tissue (AT-OB) mesenchymal stromal cells on bone tissue formation. Materials & methods: Defects were created in rat calvaria and injected with BM-OB or AT-OB and phosphate-buffered saline without cells were injected as control. Bone formation was evaluated 4 weeks postinjection. Results: Injection of BM-OB or AT-OB resulted in higher bone formation than that obtained with control. The bone tissue induced by cell injections exhibited similar mechanical properties as those of pristine calvarial bone, and its molecular cues suggested the occurrence of a remodeling process. Conclusion: Results of this study demonstrated that cell therapy with osteoblasts induced significant bone formation that exhibited the same quality as that of pre-existent bone.
- Published
- 2019
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35. The Wnt/β-catenin signaling pathway is regulated by titanium with nanotopography to induce osteoblast differentiation.
- Author
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Abuna RPF, Oliveira FS, Lopes HB, Freitas GP, Fernandes RR, Rosa AL, and Beloti MM
- Subjects
- 3T3 Cells, Animals, Cells, Cultured, Mice, Particle Size, Surface Properties, Cell Differentiation drug effects, Nanoparticles chemistry, Osteoblasts cytology, Osteoblasts drug effects, Osteogenesis drug effects, Titanium pharmacology, Wnt Signaling Pathway drug effects
- Abstract
Wnt/β-catenin signal transduction is involved in the homeostatic control of bone mass. It is well established that a titanium surface with nanotopography (Ti-Nano) favors osteoblast differentiation by modulating different signaling pathways. However, few studies have investigated the participation of the Wnt/β-catenin pathway in the osteogenic effect of nanoscale topographies. In this study, we aimed to determine whether the Wnt/β-catenin signaling pathway is involved in the elevated osteogenic potential of Ti-Nano. MC3T3-E1 cells were cultured on Ti-Nano and machined Ti (Ti-Control) for evaluation of the expression of Wnt/β-catenin signaling pathway-related genes. Based on the results to real-time PCR, the Wnt receptor Fzd4 was selected and silenced by CRISPRi. The resulting cells were cultured on both Ti surfaces, and several events involved in osteoblast differentiation were evaluated. The results revealed that Fzd4 gene silencing, corresponding to negative modulation of Wnt/β-catenin, inhibits expression of the osteoblast phenotype. It is worthy of note that this inhibitory effect on osteoblast differentiation was more pronounced in cells grown on Ti-Nano compared with those grown on Ti-Control. By disrupting Fzd4 gene expression, we have shown that the elevated osteogenic potential of Ti-Nano is due to activation of the Wnt/β-catenin signaling pathway, which reveals a new mechanism to explain osteoblast differentiation induced by nanotopography. Such an understanding of the intracellular machinery involved in surface guiding of osteoblast fate may contribute to the development of smart biomaterials to modulate the process of implant osseointegration., (Copyright © 2019 Elsevier B.V. All rights reserved.)
- Published
- 2019
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36. Titanium with nanotopography induces osteoblast differentiation through regulation of integrin αV.
- Author
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Lopes HB, Freitas GP, Fantacini DMC, Picanço-Castro V, Covas DT, Rosa AL, and Beloti MM
- Subjects
- Animals, Cell Differentiation genetics, Cell Line, Integrin alpha5 genetics, Male, Osteoblasts cytology, Rats, Rats, Wistar, Surface Properties, Cell Differentiation drug effects, Gene Expression Regulation drug effects, Integrin alpha5 biosynthesis, Nanostructures, Osteoblasts metabolism, Titanium pharmacology
- Abstract
Topographical modifications of titanium (Ti) at the nanoscale level generate surfaces that regulate several signaling pathways and cellular functions, which may affect the process of osseointegration. Here, we investigated the participation of integrin αV in the osteogenic capacity of Ti with nanotopography. Machined titanium discs (untreated) were submitted to treatment with H
2 SO4 /H2 O2 to produce the nanotopography (nanostructured). First, the greater osteogenic capacity of the nanotopography that increased osteoblast differentiation of mesenchymal stem cells compared with untreated topography was shown. Also, the nanostructured surface increased (regulation ≥ 1.9-fold) the gene expression of 6 integrins from a custom array plate utilized to evaluate the gene expression of 84 genes correlated with cell adhesion signaling pathway, including integrin αV, which is involved in osteoblast differentiation. By silencing integrin αV in MC3T3-E1 cells cultured on nanotopography, the impairment of osteoblast differentiation induced by this surface was observed. In conclusion, it was shown that nanotopography regulates the expression of several components of the cell adhesion signaling pathway and its higher osteogenic potential is, at least in part, due to its ability to upregulate the expression of integrin αV. Together with previous data that showed the participation of integrins α1, β1, and β3 in the nanotopography osseoinduction activity, we have uncovered the pivotal role of this family of membrane receptors in the osteogenic potential of this surface., (© 2019 Wiley Periodicals, Inc.)- Published
- 2019
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37. Cell Therapy: Effect of Locally Injected Mesenchymal Stromal Cells Derived from Bone Marrow or Adipose Tissue on Bone Regeneration of Rat Calvarial Defects.
- Author
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Freitas GP, Lopes HB, Souza ATP, Oliveira PGFP, Almeida ALG, Souza LEB, Coelho PG, Beloti MM, and Rosa AL
- Subjects
- Animals, Biomarkers, Bone Regeneration, Cell Differentiation, Cell Survival, Cell Tracking, Immunohistochemistry, Immunophenotyping, Male, Molecular Imaging, Osteogenesis, Rats, Treatment Outcome, X-Ray Microtomography, Cell- and Tissue-Based Therapy methods, Mesenchymal Stem Cell Transplantation methods, Mesenchymal Stem Cells cytology, Mesenchymal Stem Cells metabolism
- Abstract
Treatment of large bone defects is a challenging clinical situation that may be benefited from cell therapies based on regenerative medicine. This study was conducted to evaluate the effect of local injection of bone marrow-derived mesenchymal stromal cells (BM-MSCs) or adipose tissue-derived MSCs (AT-MSCs) on the regeneration of rat calvarial defects. BM-MSCs and AT-MSCs were characterized based on their expression of specific surface markers; cell viability was evaluated after injection with a 21-G needle. Defects measuring 5 mm that were created in rat calvaria were injected with BM-MSCs, AT-MSCs, or vehicle-phosphate-buffered saline (Control) 2 weeks post-defect creation. Cells were tracked by bioluminescence, and 4 weeks post-injection, the newly formed bone was evaluated by µCT, histology, nanoindentation, and gene expression of bone markers. BM-MSCs and AT-MSCs exhibited the characteristics of MSCs and maintained their viability after passing through the 21-G needle. Injection of both BM-MSCs and AT-MSCs resulted in increased bone formation compared to that in Control and with similar mechanical properties as those of native bone. The expression of genes associated with bone formation was higher in the newly formed bone induced by BM-MSCs, whereas the expression of genes involved in bone resorption was higher in the AT-MSC group. Cell therapy based on local injection of BM-MSCs or AT-MSCs is effective in delivering cells that induced a significant improvement in bone healing. Despite differences observed in molecular cues between BM-MSCs and AT-MSCs, both cells had the ability to induce bone tissue formation at comparable amounts and properties. These results may drive new cell therapy approaches toward complete bone regeneration.
- Published
- 2019
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38. Effect of stem cells combined with a polymer/ceramic membrane on osteoporotic bone repair.
- Author
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Almeida ALG, Freitas GP, Lopes HB, Gimenes R, Siessere S, Sousa LG, Beloti MM, and Rosa AL
- Subjects
- Animals, Barium Compounds chemistry, Bone Density, Bone Regeneration drug effects, Bone Regeneration physiology, Female, Flow Cytometry, Imaging, Three-Dimensional, Mesenchymal Stem Cells chemistry, Osteogenesis physiology, Osteoporosis physiopathology, Ovariectomy, Polyvinyls chemistry, Random Allocation, Rats, Wistar, Reproducibility of Results, Time Factors, Titanium chemistry, Treatment Outcome, Barium Compounds pharmacology, Guided Tissue Regeneration methods, Mesenchymal Stem Cells physiology, Osteogenesis drug effects, Osteoporosis therapy, Polyvinyls pharmacology, Titanium pharmacology
- Abstract
Cell therapy associated with guided bone regeneration (GBR) can be used to treat bone defects under challenging conditions such as osteoporosis. This study aimed to evaluate the effect of mesenchymal stem cells (MSCs) in combination with a poly(vinylidene-trifluoroethylene)/barium titanate (PVDF-TrFE/BT) membrane on bone repair in osteoporotic rats. Osteoporosis was induced in female rats by bilateral removal of the ovaries (OVX) or sham surgery (SHAM), and the osteoporotic condition was characterized after 5 months by microtomographic and morphometric analyses. Calvarial defects were created in osteoporotic rats that immediately received the PVDF-TrFE/BT membrane. After 2 weeks, bone marrow-derived MSCs from healthy rats, characterized by the expression of surface markers using flow cytometry, or phosphate-buffered saline (PBS) (Control) were injected into the defects and bone formation was evaluated 4 weeks post-injection by microtomographic, morphometric, and histological analyses. A reduction in the amount of bone tissue in the femurs of OVX compared with SHAM rats confirmed the osteoporotic condition of the experimental model. More bone formation was observed when the defects were injected with MSCs compared to that with PBS. The modification that we are proposing in this study for the classical GBR approach where cells are locally injected after a membrane implantation may be a promising therapeutic strategy to increase bone formation under osteoporotic condition.
- Published
- 2019
- Full Text
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39. Effect of cell source and osteoblast differentiation on gene expression profiles of mesenchymal stem cells derived from bone marrow or adipose tissue.
- Author
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Fideles SOM, Ortiz AC, Assis AF, Duarte MJ, Oliveira FS, Passos GA, Beloti MM, and Rosa AL
- Abstract
Mesenchymal stem cells (MSCs) have been used in therapies for bone tissue healing. The aim of this study was to investigate the effect of cell source and osteoblast differentiation on gene expression profiles of MSCs from bone marrow (BM-MSCs) or adipose tissue (AT-MSCs) to contribute for selecting a suitable cell population to be used in cell-based strategies for bone regeneration. BM-MSCs and AT-MSCs were cultured in growth medium to keep MSCs characteristics or in osteogenic medium to induce osteoblast differentiation (BM-OBs and AT-OBs). The transcriptomic analysis was performed by microarray covering the entire rat functional genome. It was observed that cells from bone marrow presented higher expression of genes related to osteogenesis, whereas cells from adipose tissue showed a higher expression of genes related to angiogenesis and adipocyte differentiation, irrespective of cell differentiation. By comparing cells from the same source, MSCs from both sources exhibited higher expression of genes involved in angiogenesis, osteoblast differentiation, and bone morphogenesis than osteoblasts. The clustering analysis showed that AT-OBs exhibited a gene expression profile closer to MSCs from both sources than BM-OBs, suggesting that BM-OBs were in a more advanced stage of differentiation. In conclusion, our results suggest that in cell-based therapies for bone regeneration AT-MSCs could be considered for angiogenic purposes, whereas BM-MSCs and osteoblasts differentiated from either source could be better for osteogenic approaches., (© 2019 Wiley Periodicals, Inc.)
- Published
- 2019
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40. Participation of integrin β3 in osteoblast differentiation induced by titanium with nano or microtopography.
- Author
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Lopes HB, Freitas GP, Elias CN, Tye C, Stein JL, Stein GS, Lian JB, Rosa AL, and Beloti MM
- Subjects
- Animals, Cell Line, Male, Mice, Osteoblasts cytology, Rats, Rats, Wistar, Cell Differentiation, Integrin beta3 metabolism, Nanostructures chemistry, Osteoblasts metabolism, Titanium chemistry, Wnt Signaling Pathway
- Abstract
The major role of integrins is to mediate cell adhesion but some of them are involved in the osteoblasts-titanium (Ti) interactions. In this study, we investigated the participation of integrins in osteoblast differentiation induced by Ti with nanotopography (Ti-Nano) and with microtopography (Ti-Micro). By using a PCR array, we observed that, compared with Ti-Micro, Ti-Nano upregulated the expression of five integrins in mesenchymal stem cells, including integrin β3, which increases osteoblast differentiation. Silencing integrin β3, using CRISPR-Cas9, in MC3T3-E1 cells significantly reduced the osteoblast differentiation induced by Ti-Nano in contrast to the effect on T-Micro. Concomitantly, integrin β3 silencing downregulated the expression of integrin αv, the parent chain that combines with other integrins and several components of the Wnt/β-catenin and BMP/Smad signaling pathways, all involved in osteoblast differentiation, only in cells cultured on Ti-Nano. Taken together, our results showed the key role of integrin β3 in the osteogenic potential of Ti-Nano but not of Ti-Micro. Additionally, we propose a novel mechanism to explain the higher osteoblast differentiation induced by Ti-Nano that involves an intricate regulatory network triggered by integrin β3 upregulation, which activates the Wnt and BMP signal transductions. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1303-1313, 2019., (© 2019 Wiley Periodicals, Inc.)
- Published
- 2019
- Full Text
- View/download PDF
41. The microRNA-23a cluster regulates the developmental HoxA cluster function during osteoblast differentiation.
- Author
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Godfrey TC, Wildman BJ, Beloti MM, Kemper AG, Ferraz EP, Roy B, Rehan M, Afreen LH, Kim E, Lengner CJ, and Hassan Q
- Subjects
- Acetylation, Animals, Core Binding Factor Alpha 1 Subunit genetics, Core Binding Factor Alpha 1 Subunit metabolism, HEK293 Cells, Histones genetics, Histones metabolism, Homeodomain Proteins genetics, Humans, Mice, MicroRNAs genetics, Osteoblasts cytology, Osteogenesis, Phosphoproteins genetics, Transcription Factors, 3' Untranslated Regions, Cell Differentiation, Homeodomain Proteins metabolism, MicroRNAs metabolism, Multigene Family, Osteoblasts metabolism, Phosphoproteins metabolism
- Abstract
MicroRNAs (miRs) and Hox transcription factors have decisive roles in postnatal bone formation and homeostasis. In silico analysis identified extensive interaction between HOXA cluster mRNA and microRNAs from the miR-23a cluster. However, Hox regulation by the miR-23a cluster during osteoblast differentiation remains undefined. We examined this regulation in preosteoblasts and in a novel miR-23a cluster knockdown mouse model. Overexpression and knockdown of the miR-23a cluster in preosteoblasts decreased and increased, respectively, the expression of the proteins HOXA5, HOXA10, and HOXA11; these proteins' mRNAs exhibited significant binding with the miR-23a cluster miRNAs, and miRNA 3'-UTR reporter assays confirmed repression. Importantly, during periods correlating with development and differentiation of bone cells, we found an inverse pattern of expression between HoxA factors and members of the miR-23a cluster. HOXA5 and HOXA11 bound to bone-specific promoters, physically interacted with transcription factor RUNX2, and regulated bone-specific genes. Depletion of HOXA5 or HOXA11 in preosteoblasts also decreased cellular differentiation. Additionally, stable overexpression of the miR-23a cluster in osteoblasts decreased the recruitment of HOXA5 and HOXA11 to osteoblast gene promoters, significantly inhibiting histone H3 acetylation. Heterozygous miR-23a cluster knockdown female mice (miR-23a Cl
WT/ZIP ) had significantly increased trabecular bone mass when compared with WT mice. Furthermore, miR-23a cluster knockdown in calvarial osteoblasts of these mice increased the recruitment of HOXA5 and HOXA11, with a substantial enrichment of promoter histone H3 acetylation. Taken together, these findings demonstrate that the miR-23a cluster is required for maintaining stage-specific HoxA factor expression during osteogenesis., (© 2018 Godfrey et al.)- Published
- 2018
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42. Effect of bone morphogenetic protein 9 on osteoblast differentiation of cells grown on titanium with nanotopography.
- Author
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Souza ATP, Bezerra BLS, Oliveira FS, Freitas GP, Bighetti Trevisan RL, Oliveira PT, Rosa AL, and Beloti MM
- Subjects
- Adaptor Proteins, Signal Transducing genetics, Analysis of Variance, Animals, Cell Adhesion physiology, Cell Line, Core Binding Factor Alpha 1 Subunit genetics, Gene Expression, Membrane Proteins genetics, Mice, Osteoblasts drug effects, Osteogenesis drug effects, Smad4 Protein metabolism, Smad6 Protein metabolism, Surface Properties, Cell Differentiation drug effects, Growth Differentiation Factor 2 pharmacology, Nanopores ultrastructure, Osteoblasts cytology, Titanium chemistry, Titanium metabolism
- Abstract
Among bone morphogenetic proteins (BMPs), BMP-9 has been described as one with higher osteogenic potential. Here, we aimed at evaluating the effect of BMP-9 on the osteoblast differentiation of cells grown on titanium (Ti) with nanotopography, a well-known osseoinductive surface. MC3T3-E1 cells were grown either in absence or presence of BMP-9 (20 nM) on Ti with nanotopography (Ti-Nano) or machined Ti (Ti-Machined) for up to 21 days to evaluate the gene expression of RUNX2, osterix, osteocalcin, bone sialoprotein, SMAD6 and SMAD4, protein expression of SMAD4, ALP activity and extracellular matrix mineralization. As expected BMP-9 increased osteoblast differentiation irrespective of Ti surface topography; however, the cells grown on Ti-Nano were more responsible to BMP-9 compared with cells grown on Ti-machined. This could be, at least in part, due to the fact that Ti-Nano may act on both ways, by increasing the activation (SMAD4) and decreasing the inhibition (SMAD6) of the signaling pathway triggered by BMP-9, while Ti-Machined only decrease the inhibition (SMAD6) of this pathway. In conclusion, the combination of the osteogenic potential of BMP-9 with the osseoinductive capacity of Ti-Nano could be a promising strategy to favor the osseointegration of Ti implants., (© 2018 Wiley Periodicals, Inc.)
- Published
- 2018
- Full Text
- View/download PDF
43. Effect of cell therapy with allogeneic osteoblasts on bone repair of rat calvaria defects.
- Author
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Souza ATP, Freitas GP, Lopes HB, Ferraz EP, Oliveira FS, Beloti MM, and Rosa AL
- Subjects
- Alkaline Phosphatase metabolism, Animals, Biomarkers, Cells, Cultured, Extracellular Matrix metabolism, Gene Expression Regulation, Osteoblasts metabolism, Osteoblasts physiology, Osteocalcin biosynthesis, Osteocalcin genetics, Osteogenesis physiology, Rats, Wistar, Skull cytology, Transplantation, Homologous methods, X-Ray Microtomography, Cell- and Tissue-Based Therapy methods, Osteoblasts transplantation, Skull injuries
- Abstract
Background Aims: Regenerative medicine strategies based on cell therapy are considered a promising approach to repair bone defects. The aims of this study were to evaluate the effect of subculturing on the osteogenic potential of osteoblasts derived from newborn rat calvaria and the effect of these osteoblasts on bone repair of rat calvaria defects., Methods: Cells were obtained from 50 newborn rat calvaria, and primary osteoblasts (OB) were compared with first passage (OB-P1) in terms of osteogenic potential by assaying cell proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization and gene expression of the osteoblastic markers RUNX2, ALP, osteocalcin and bone sialoprotein. Then, 5-mm calvaria defects were created in 24 Wistar rats, and after 2 weeks, they were locally injected with 50 µL of phosphate-buffered saline containing either 5 × 10
6 osteoblasts (OB-P1, n = 12) or no cells (control, n = 12). Four weeks post-injection, the bone formation was evaluated by micro-computed tomography and histological analyses. Data were compared by analysis of variance, followed by the Student-Newman-Keuls's test or Student's t-test (P ≤ 0.05)., Results: OB-P1 showed high proliferation and ALP activity, and despite the reduced gene expression of osteoblastic markers and extracellular matrix mineralization compared with OB, they displayed osteogenic potential, being a good choice for injection into calvaria defects. The micro-tomographic and histological data showed that defects treated with OB-P1 presented higher bone formation compared with control defects., Discussion: Our results indicate that cells derived from newborn rat calvaria retain osteoblastic characteristics after subculturing and that these osteoblasts stimulate bone repair in a rat calvaria defect model., (Copyright © 2018 Elsevier Ltd. All rights reserved.)- Published
- 2018
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44. Selection of reference genes for quantitative real-time polymerase chain reaction studies in rat osteoblasts.
- Author
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Abuna RPF, Oliveira FS, Ramos JIR, Lopes HB, Freitas GP, Souza ATP, Beloti MM, and Rosa AL
- Subjects
- Animals, Animals, Newborn, Cell Line, Core Binding Factor Alpha 1 Subunit genetics, Core Binding Factor Alpha 1 Subunit metabolism, Endopeptidases genetics, Endopeptidases metabolism, Mesenchymal Stem Cells cytology, Osteoblasts metabolism, Rats, Real-Time Polymerase Chain Reaction, Reference Standards, Gene Expression genetics, Gene Expression Profiling methods, Mesenchymal Stem Cells metabolism, Osteoblasts cytology
- Abstract
Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool to evaluate gene expression, but its accuracy depends on the choice and stability of the reference genes used for normalization. In this study, we aimed to identify reference genes for studies on osteoblasts derived from rat bone marrow mesenchymal stem cells (bone marrow osteoblasts), osteoblasts derived from newborn rat calvarial (calvarial osteoblasts), and rat osteosarcoma cell line UMR-106. The osteoblast phenotype was characterized by ALP activity and extracellular matrix mineralization. Thirty-one candidates for reference genes from a Taqman
® array were assessed by qRT-PCR, and their expressions were analyzed by five different approaches. The data showed that several of the most traditional reference genes, such as Actb and Gapdh, were inadequate for normalization and that the experimental conditions may affect gene stability. Eif2b1 was frequently identified among the best reference genes in bone marrow osteoblasts, calvarial osteoblasts, and UMR-106 osteoblasts. Selected stable and unstable reference genes were used to normalize the gene expression of Runx2, Alp, and Oc. The data showed statistically significant differences in the expression of these genes depending on the stability of the reference gene used for normalization, creating a bias that may induce incorrect assumptions in terms of osteoblast characterization of these cells. In conclusion, our study indicates that a rigorous selection of reference genes is a key step in qRT-PCR studies in osteoblasts to generate precise and reliable data., (© 2018 Wiley Periodicals, Inc.)- Published
- 2018
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45. The effect of collagen coating on titanium with nanotopography on in vitro osteogenesis.
- Author
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Costa DG, Ferraz EP, Abuna RPF, de Oliveira PT, Morra M, Beloti MM, and Rosa AL
- Subjects
- Animals, Cell Differentiation, Cell Proliferation, Cells, Cultured, Osteoblasts metabolism, Rats, Rats, Wistar, Surface Properties, Coated Materials, Biocompatible chemistry, Collagen chemistry, Nanostructures chemistry, Osteoblasts cytology, Osteogenesis, Titanium chemistry
- Abstract
Several studies have shown the positive effects of Ti either with nanotopography or coated with collagen on osteoblast differentiation. Thus, we hypothesized that the association of nanotopography with collagen may increase the in vitro osteogenesis on Ti surface. Ti discs with nanotopography with or without collagen coating were characterized by scanning electron microscopy and atomic force microscopy. Rat calvaria-derived osteoblastic cells were cultured on both Ti surfaces for up to 14 days and the following parameters were evaluated: cell proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization, protein expression of bone sialoprotein (BSP) and osteopontin (OPN), and gene expression of collagen type 1a (Coll1a), runt-related transcription factor 2 (Runx2), osterix (OSX), osteocalcin (OC), Ki67, Survivin, and Bcl2-associated X protein (BAX). Surface characterization evidenced that collagen coating did not alter the nanotopography. Collagen coating increased cell proliferation, ALP activity, extracellular matrix mineralization, and Coll1a, OSX, OC, and BAX gene expression. Also, OPN and BSP proteins were strongly detected in cultures grown on both Ti surfaces. In conclusion, our results showed that the combination of nanotopography with collagen coating stimulates the early, intermediate, and final events of the in vitro osteogenesis and may be considered a potential approach to promote osseointegration of Ti implants. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2783-2788, 2017., (© 2017 Wiley Periodicals, Inc.)
- Published
- 2017
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46. Participation of extracellular signal-regulated kinases 1/2 in osteoblast and adipocyte differentiation of mesenchymal stem cells grown on titanium surfaces.
- Author
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Silva HF, Abuna RPF, Lopes HB, Francischini MS, de Oliveira PT, Rosa AL, and Beloti MM
- Subjects
- Animals, Cells, Cultured, Flavonoids pharmacology, Gene Expression, Male, Microscopy, Electron, Scanning, Mitogen-Activated Protein Kinase 3 antagonists & inhibitors, Rats, Rats, Wistar, Real-Time Polymerase Chain Reaction, Surface Properties, Adipocytes metabolism, Cell Differentiation drug effects, Mesenchymal Stem Cells metabolism, Mitogen-Activated Protein Kinase 3 pharmacology, Osteoblasts metabolism, Titanium chemistry
- Abstract
Osteoblasts and adipocytes coexist in the implantation site and affect the process of titanium (Ti) osseointegration. As extracellular signal-regulated kinases 1/2 (ERK1/2) are involved in osteogenesis and adipogenesis, the aim of our study was to investigate if the effects of Ti surface topography on osteoblast and adipocyte differentiation are modulated by ERK1/2. The experiments were conducted based on the effect of the ERK1/2 inhibitor, PD98059, on mesenchymal stem cells (MSCs) grown under osteogenic and adipogenic conditions on Ti with nanotopography (Ti-Nano) or on machined Ti (Ti-Machined). The results showed that, in general, ERK1/2 inhibition favored osteoblast and adipocyte differentiation of MSCs grown on Ti-Machined. In MSCs grown on Ti-Nano, ERK1/2 inhibition upregulated the expression of alkaline phosphatase and osteocalcin and reduced extracellular matrix mineralization. In terms of adipocyte differentiation, ERK1/2 inhibition elicited similar MSC responses to Ti-Nano and Ti-Machined, upregulating gene expression of adipocyte markers without affecting lipid accumulation. Our results indicate that, under osteogenic and adipogenic conditions, the responses of MSCs to Ti surface topography in terms of osteogenesis and adipogenesis are dependent on ERK1/2. Thus, a precise modulation of ERK1/2 expression and activity induced by surface topography could be a good strategy to drive the process of implant osseointegration., (© 2017 Eur J Oral Sci.)
- Published
- 2017
- Full Text
- View/download PDF
47. Potential of Osteoblastic Cells Derived from Bone Marrow and Adipose Tissue Associated with a Polymer/Ceramic Composite to Repair Bone Tissue.
- Author
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Freitas GP, Lopes HB, Almeida ALG, Abuna RPF, Gimenes R, Souza LEB, Covas DT, Beloti MM, and Rosa AL
- Subjects
- Adipose Tissue cytology, Animals, Barium Compounds, Biocompatible Materials, Bone Marrow Cells cytology, Cell Differentiation, Male, Mesenchymal Stem Cells cytology, Polyvinyls, Rats, Rats, Wistar, Titanium, Mesenchymal Stem Cell Transplantation methods, Osteoblasts cytology, Skull, Tissue Engineering methods
- Abstract
One of the tissue engineering strategies to promote bone regeneration is the association of cells and biomaterials. In this context, the aim of this study was to evaluate if cell source, either from bone marrow or adipose tissue, affects bone repair induced by osteoblastic cells associated with a membrane of poly(vinylidene-trifluoroethylene)/barium titanate (PVDF-TrFE/BT). Mesenchymal stem cells (MSC) were isolated from rat bone marrow and adipose tissue and characterized by detection of several surface markers. Also, both cell populations were cultured under osteogenic conditions and it was observed that MSC from bone marrow were more osteogenic than MSC from adipose tissue. The bone repair was evaluated in rat calvarial defects implanted with PVDF-TrFE/BT membrane and locally injected with (1) osteoblastic cells differentiated from MSC from bone marrow, (2) osteoblastic cells differentiated from MSC from adipose tissue or (3) phosphate-buffered saline. Luciferase-expressing osteoblastic cells derived from bone marrow and adipose tissue were detected in bone defects after cell injection during 25 days without difference in luciferin signal between cells from both sources. Corroborating the in vitro findings, osteoblastic cells from bone marrow combined with the PVDF-TrFE/BT membrane increased the bone formation, whereas osteoblastic cells from adipose tissue did not enhance the bone repair induced by the membrane itself. Based on these findings, it is possible to conclude that, by combining a membrane with cells in this rat model, cell source matters and that bone marrow could be a more suitable source of cells for therapies to engineer bone.
- Published
- 2017
- Full Text
- View/download PDF
48. Bioactive glass-based surfaces induce differential gene expression profiling of osteoblasts.
- Author
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Ferraz EP, Oliveira FS, de Oliveira PT, Crovace MC, Peitl-Filho O, Beloti MM, and Rosa AL
- Subjects
- Animals, Gene Expression Profiling, Male, Osteoblasts cytology, Rats, Rats, Wistar, Surface Properties, Calcification, Physiologic drug effects, Ceramics pharmacology, Gene Expression Regulation drug effects, Osteoblasts metabolism
- Abstract
The ability of Biosilicate® with two crystalline phases (BioS-2P) to drive osteoblast differentiation encourages the investigation of the cellular mechanisms involved in this process. Then, the aim of our study was to analyze the large-scale gene expression of osteoblasts grown on BioS-2P compared with Bioglass
® 45S5 (45S5). Osteoblasts differentiated from rat bone marrow mesenchymal stem cells were cultured under osteogenic conditions on BioS-2P, 45S5 and polystyrene (control). After 10 days, the expression of 23,794 genes was analyzed using mRNA Sequencing and the data were validated by real-time PCR. The BioS-2P exhibited 5 genes upregulated and 3 downregulated compared with 45S5. Compared with control, BioS-2P upregulated 15 and downregulated 11 genes, while 45S5 upregulated 25 and downregulated 21 genes. Eight genes were commonly upregulated and 4 downregulated by both bioactive glasses. In conclusion, our results demonstrated that bioactive glasses affect the gene expression profiling of osteoblasts. Most of the regulated genes by both BioS-2P and 45S5 are associated with the process of mineralization highlighting their osteostimulation property that is, at least in part, derived from the ability to modulate the intracellular machinery to promote osteoblast genotype expression. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 419-423, 2017., (© 2016 Wiley Periodicals, Inc.)- Published
- 2017
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49. Poly(Vinylidene Fluoride-Trifluorethylene)/barium titanate membrane promotes de novo bone formation and may modulate gene expression in osteoporotic rat model.
- Author
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Scalize PH, Bombonato-Prado KF, de Sousa LG, Rosa AL, Beloti MM, Semprini M, Gimenes R, de Almeida AL, de Oliveira FS, Hallak Regalo SC, and Siessere S
- Subjects
- Animals, Biocompatible Materials chemistry, Bone Regeneration, Bone Transplantation, Bone and Bones metabolism, Cathepsin K metabolism, Disease Models, Animal, Female, Gene Expression Profiling, Gene Expression Regulation, Matrix Metalloproteinase 9 metabolism, Membranes, Artificial, Osteoblasts metabolism, Osteoclasts metabolism, Osteoporosis metabolism, RANK Ligand metabolism, Rats, Rats, Wistar, Real-Time Polymerase Chain Reaction, Receptor Activator of Nuclear Factor-kappa B metabolism, Receptors, Calcitonin metabolism, X-Ray Microtomography, Barium Compounds chemistry, Hydrocarbons, Fluorinated chemistry, Osteogenesis, Osteoporosis surgery, Titanium chemistry, Vinyl Compounds chemistry
- Abstract
Osteoporosis is a chronic disease that impairs proper bone remodeling. Guided bone regeneration is a surgical technique that improves bone defect in a particular region through new bone formation, using barrier materials (e.g. membranes) to protect the space adjacent to the bone defect. The polytetrafluorethylene membrane is widely used in guided bone regeneration, however, new membranes are being investigated. The purpose of this study was to evaluate the effect of P(VDFTrFE)/BT [poly(vinylidene fluoride-trifluoroethylene)/barium titanate] membrane on in vivo bone formation. Twenty-three Wistar rats were submitted to bilateral ovariectomy. Five animals were subjected to sham surgery. After 150 days, bone defects were created and filled with P(VDF-TrFE)/BT membrane or PTFE membrane (except for the sham and OVX groups). After 4 weeks, the animals were euthanized and calvaria samples were subjected to histomorphometric and computed microtomography analysis (microCT), besides real time polymerase chain reaction (real time PCR) to evaluate gene expression. The histomorphometric analysis showed that the animals that received the P(VDF-TrFE)/BT membrane presented morphometric parameters similar or even better compared to the animals that received the PTFE membrane. The comparison between groups showed that gene expression of RUNX2, BSP, OPN, OSX and RANKL were lower on P(VDF-TrFE)/BT membrane; the gene expression of ALP, OC, RANK and CTSK were similar and the gene expression of OPG, CALCR and MMP9 were higher when compared to PTFE. The results showed that the P(VDF-TrFE)/BT membrane favors bone formation, and therefore, may be considered a promising biomaterial to support bone repair in a situation of osteoporosis.
- Published
- 2016
- Full Text
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50. Participation of MicroRNA-34a and RANKL on bone repair induced by poly(vinylidene-trifluoroethylene)/barium titanate membrane.
- Author
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Lopes HB, Ferraz EP, Almeida AL, Florio P, Gimenes R, Rosa AL, and Beloti MM
- Subjects
- Animals, Bone Marrow Cells cytology, Bone Marrow Cells metabolism, Bone and Bones metabolism, Cell Differentiation, Gene Expression, Membranes, Artificial, Osteoblasts metabolism, Osteogenesis, Rats, Wistar, Barium Compounds chemistry, Bone Regeneration, Hydrocarbons, Fluorinated chemistry, MicroRNAs metabolism, Osteoblasts cytology, RANK Ligand metabolism, Titanium chemistry, Vinyl Compounds chemistry
- Abstract
The poly(vinylidene-trifluoroethylene)/barium titanate (PVDF) membrane enhances in vitro osteoblast differentiation and in vivo bone repair. Here, we hypothesized that this higher bone repair could be also due to bone resorption inhibition mediated by a microRNA (miR)/RANKL circuit. To test our hypothesis, the large-scale miR expression of bone tissue grown on PVDF and polytetrafluoroethylene (PTFE) membranes was evaluated to identify potential RANKL-targeted miRs modulated by PVDF. The animal model used was rat calvarial defects implanted with either PVDF or PTFE. At 4 and 8 weeks, the bone tissue grown on membranes was submitted to a large-scale analysis of miRs by microarray. The expression of miR-34a and some of its targets, including RANKL, were evaluated by real-time polimerase chain reaction and osteoclast activity was detected by tartrate-resistant acid phosphatase (TRAP) staining. Among more than 250 miRs, twelve, including miR-34a, were simultaneously higher expressed (≥2 fold) at 4 and 8 weeks on PVDF. The higher expression of miR-34a was concomitant with a reduced expression of all its evaluated targets, including RANKL. Additionally, more TRAP-positive cells were observed in bone tissue grown on PTFE compared with PVDF in both time points. In conclusion, our results suggest that the higher bone formation induced by PVDF could be, at least in part, triggered by a miR-34a increase and RANKL decrease, which may inhibit osteoclast differentiation and activity, and bone resorption.
- Published
- 2016
- Full Text
- View/download PDF
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