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1.
Molecules ; 26(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34072168

RESUMO

This study aims to investigate whether the in vitro-cultured L. pumila var. alata has higher antioxidant activity than its wild plant. An 8-week-old L. pumila var. alata nodal segment and leaf explants were cultured onto Murashige and Skoog (MS) medium supplemented with various cytokinins (zeatin, kinetin, and 6-benzylaminopurine (BAP)) for shoot multiplication and auxins (2,4-dichlorophenoxyacetic acid (2,4-D) and picloram) for callus induction, respectively. The results showed that 2 mg/L zeatin produced the optimal results for shoot and leaf development, and 0.5 mg/L 2,4-D produced the highest callus induction results (60%). After this, 0.5 mg/L 2,4-D was combined with 0.25 mg/L cytokinins and supplemented to the MS medium. The optimal results for callus induction (100%) with yellowish to greenish and compact texture were obtained using 0.5 mg/L 2,4-D combined with 0.25 mg/L zeatin. Leaves obtained from in vitro plantlets and wild plants as well as callus were extracted and analyzed for their antioxidant activities (DPPH and FRAP methods) and polyphenolic properties (total flavonoid and total phenolic content). When compared with leaf extracts of in vitro plantlets and wild plants of L. pumila var. alata, the callus extract displayed significantly higher antioxidant activities and total phenolic and flavonoid content. Hence, callus culture potentially can be adapted for antioxidant and polyphenolic production to satisfy pharmaceutical and nutraceutical needs while conserving wild L. pumila var. alata.


Assuntos
Calo Ósseo/efeitos dos fármacos , Reguladores de Crescimento de Plantas/metabolismo , Brotos de Planta/efeitos dos fármacos , Polifenóis/química , Primulaceae/efeitos dos fármacos , Ácido 2,4-Diclorofenoxiacético/química , Antioxidantes/química , Compostos de Benzil/química , Compostos de Bifenilo/química , Meios de Cultura , Suplementos Nutricionais , Flavonoides/química , Técnicas In Vitro , Cinetina/química , Fenol/química , Picloram/química , Picratos/química , Folhas de Planta , Proteínas de Plantas , Raízes de Plantas/efeitos dos fármacos , Plantas/efeitos dos fármacos , Purinas/química , Zeatina/química
2.
Eur Cell Mater ; 41: 592-602, 2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-34027631

RESUMO

Calcium channel blockers (CCBs), which are widely used in the treatment of hypertension, have been shown to influence bone metabolism. However, there is little information on whether CCBs also influence the process of fracture healing. Therefore, the effect of the CCB amlodipine on bone healing was studied in a stable closed fracture model in mice using intramedullary screw fixation. Bone healing was investigated by radiology, biomechanics, histomorphometry and Western blot analysis 2 and 5 weeks after fracture healing. Animals were treated daily (post operatively) per os using a gavage with amlodipine low dose (1 mg/ kg body weight, n = 20), amlodipine high dose (3 mg/kg body weight, n = 20) or vehicle (NaCl) (control, n = 20) serving as a negative control. At 2 and 5 weeks, histomorphometric analysis revealed a significantly larger amount of bone tissue within the callus of amlodipine low-dose- and high-dose-treated animals when compared to controls. This was associated with a smaller amount of cartilaginous and fibrous tissue, indicating an acceleration of fracture healing. Biomechanics showed a slightly, but not significantly, higher bending stiffness in amlodipine low-dose- and high-dose-treated animals. Western blot analysis revealed a significantly increased expression of bone morphogenetic protein (BMP)-2 and vascular endothelial growth factor (VEGF). Moreover, the analysis showed a 5-fold higher expression of osteoprotegerin (OPG) and a 10-fold elevated expression of the receptor activator of NF-κB ligand (RANKL), indicating an increased bone turnover. These findings demonstrated that amlodipine accelerated fracture healing by stimulating bone formation, callus remodelling and osteoclast activity.


Assuntos
Anlodipino/farmacologia , Fraturas do Fêmur/tratamento farmacológico , Fêmur/efeitos dos fármacos , Consolidação da Fratura/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 2/metabolismo , Remodelação Óssea/efeitos dos fármacos , Parafusos Ósseos , Calo Ósseo/efeitos dos fármacos , Calo Ósseo/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Modelos Animais de Doenças , Fraturas do Fêmur/metabolismo , Fêmur/metabolismo , Camundongos , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Ligante RANK/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
3.
Mol Biol Rep ; 47(9): 6887-6897, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32914263

RESUMO

The objective of the present work was the selection of cultivar, suitable medium and explant type for callus, root production, ascorbic acid, total ascorbic acid, dehydroascorbic and total protein of non-heading Chinese cabbage in two cultivars 'Caixin' and 'Suzhouqing'. We compared 10 types of MS media supplemented with 0.0, 1.0, 2.0 and 3.0 mg/l TDZ; 0.0, 0.25, 0.50 and 1.0 mg/l NAA and 0.0, 5.0, 7.5 and 9.0 mg/l AgNO3 and 5 kinds of explants as embryo, leaf, root, cotyledon and hypocotyl. Maximum frequency of callus fresh weight was recorded with hypocotyl explant, which were cultured on MS + 2.0 mg/l TDZ + 1.0 mg/l NAA + 9.0 mg/l AgNO3 in 'Suzhouqing', optimum callus dry weight was obtained on the same media. The highest result for root fresh and dry weight recorded with 'Caixin' with MS + 3.0 mg/l TDZ + 1.0 mg/l NAA + 9.0 mg/l AgNO3 when we used embryo as explant. The highest ascorbic acid content was found with callus cultured on MS + 1.0 mg/l TDZ + 0.25 mg/l NAA + 5.0 mg/l AgNO3, when used leaf explant in 'Caixin' or root in 'Suzhouqing', and there were no significant difference between them. While the highest value of total AsA content was registered with callus cultured on MS + 2.0 mg/l TDZ + 0.25 mg/l NAA + 5.0 mg/l AgNO3 extracted from cotyledon in 'Caixin'. The highest content of DHA was registered with MS + 2.0 mg/l TDZ + 0.25 mg/l NAA + 5.0 mg/l AgNO3 with cotyledon in 'Caixin'. Also, in 'Caixin' MS + 3.0 mg/l TDZ + 0.25 mg/l NAA + 5.0 mg/l AgNO3 recorded the highest value of total protein content with embryo explant.


Assuntos
Ácido Ascórbico/análise , Calo Ósseo/efeitos dos fármacos , Brassica rapa/metabolismo , Técnicas de Cultura de Células/métodos , Reguladores de Crescimento de Plantas/farmacologia , Raízes de Plantas/efeitos dos fármacos , Proteínas/análise , Ácido Ascórbico/análogos & derivados , Ácido Ascórbico/metabolismo , Calo Ósseo/crescimento & desenvolvimento , Calo Ósseo/metabolismo , Brassica rapa/crescimento & desenvolvimento , Células Cultivadas , Naftalenos/farmacologia , Compostos de Fenilureia/farmacologia , Células Vegetais/efeitos dos fármacos , Células Vegetais/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Nitrato de Prata/farmacologia , Tiadiazóis/farmacologia
4.
Eur Cell Mater ; 39: 183-192, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32195554

RESUMO

Around 10 % of long bone fractures show inadequate bone healing resulting in non-union development. A deregulated arginine-citrulline-nitric oxide metabolism caused by a poor nutritional status of the patients is a risk factor for non-unions. Additionally, previous research in mice with a disrupted arginine to citrulline conversion showed delayed healing. The study hypothesis was that stimulating said metabolism could positively influence the healing process through promotion of collagen synthesis and angiogenesis. Adult wild-type mice underwent a femur osteotomy and plate-screw osteosynthesis. Mice were randomly divided into three groups and received daily oral supplementation of arginine, citrulline or 0.9 % saline (control). Body weight and food intake were measured daily. After 14 d, the mice were euthanised and femora collected. Callus formation was assessed by micro-computed tomography and concentrations of amino acids and enzymes in the femora were measured. Only citrulline-treated mice showed significantly increased bridging of the fracture gap when compared to control mice. Femur citrulline and ornithine concentrations were increased in citrulline-treated animals. qPCR showed significantly decreased expression of inflammatory markers, whereas increased expression of angiogenic and collagen-producing factors was observed in citrulline-treated mice. Although food intake did not show any difference between the three groups, animals treated with citrulline showed a weight gain of 0.3 g, compared with a 0.1 g decline in the control group. Daily oral citrulline supplementation stimulated callus formation and improved the inflammatory response, positively contributing to the enhanced healing response. Finally, the increased weight gain pointed toward a better post-operative recovery.


Assuntos
Citrulina/farmacologia , Suplementos Nutricionais , Consolidação da Fratura/efeitos dos fármacos , Aminoácidos/análise , Animais , Peso Corporal/efeitos dos fármacos , Calo Ósseo/diagnóstico por imagem , Calo Ósseo/efeitos dos fármacos , Comportamento Alimentar/efeitos dos fármacos , Feminino , Fêmur/metabolismo , Imageamento Tridimensional , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
5.
Calcif Tissue Int ; 106(2): 172-179, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31578632

RESUMO

Clinical concerns have been raised over prior exposure to bisphosphonates impairing fracture healing. To model this, groups of male Wistar rats were assigned to saline control or treatment groups receiving 0.15 mg/kg (low dose), 0.5 mg/kg (medium dose), and 5 mg/kg (high dose) Pamidronate (PAM) twice weekly for 4 weeks. At this point, closed fractures were made using an Einhorn apparatus, and bisphosphonate dosing was continued until the experimental endpoint. Specimens were analyzed at 2 and 6 weeks (N = 8 per group per time point). Twice weekly PAM dosing was found to have no effect on early soft callus remodeling at 2 weeks post fracture. At this time point, the highest dose PAM group gave significant increases in bone volume (+ 10%, p < 0.05), bone mineral content (+ 30%, p < 0.01), and bone mineral density (+ 10%, p < 0.01). This PAM dosing regimen showed more substantive effects on hard callus at 6 weeks post fracture, with PAM treatment groups showing + 46-79% increased bone volume. Dynamic bone labeling showed reduced calcein signal in the PAM-treated calluses (38-63%, p < 0.01) and reduced MAR (32-49%, p < 0.01), suggesting a compensatory reduction in bone anabolism. These data support the concept that bisphosphonates lead to profound decreases in bone turnover in fracture repair, however, this does not affect soft callus remodeling.


Assuntos
Calo Ósseo/efeitos dos fármacos , Fraturas do Fêmur/patologia , Fraturas Fechadas/patologia , Osteogênese/efeitos dos fármacos , Pamidronato/farmacologia , Animais , Densidade Óssea/efeitos dos fármacos , Conservadores da Densidade Óssea/administração & dosagem , Conservadores da Densidade Óssea/farmacologia , Remodelação Óssea/efeitos dos fármacos , Calo Ósseo/patologia , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Fraturas do Fêmur/tratamento farmacológico , Consolidação da Fratura/efeitos dos fármacos , Fraturas Fechadas/tratamento farmacológico , Masculino , Tamanho do Órgão/efeitos dos fármacos , Osteogênese Imperfeita/patologia , Pamidronato/administração & dosagem , Ratos , Ratos Wistar , Fatores de Tempo
6.
J Orthop Surg Res ; 14(1): 206, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31277691

RESUMO

BACKGROUND: The positive effects of grape seed proanthocyanidin extract (GSPE) on bone health, which is a potent antioxidant, are known but its effects on fracture healing are not sufficiently covered in the literature. This study aims to investigate the effects of GSPE on fracture healing and biomechanics of healing bone. MATERIALS AND METHODS: Sixty-four adult Wistar-Albino male rats were divided into 8 groups of 8 animals in each group. Osteotomy was performed to the right femurs of all groups except the negative control (G1) and positive control (G2) groups, and intramedullary Kirchner wire was used for fixation. GSPE was given to half of the rats (G2-G4-G6-G8) 100 mg/kg/day by oral gavage. The rats were sacrificed on the tenth (G3-G4), twentieth (G5-G6), and thirtieth (G1-G2-G7-G8) days, respectively, and histopathological, radiological, and biomechanical examinations were performed. RESULTS: Histopathological examination of the specimens from the callus tissues revealed that bone healing was more prominent in the groups supplemented with GSPE (G4, G6, G8). There was a statistically significant improvement in radiological recovery scores and callus volumes in groups with GSPE. When biomechanical strengths were evaluated, it was found that GSPE increased bone strength not only in fracture groups but also in the positive control group (G2). CONCLUSIONS: As a result, this study showed that GSPE, a potent anti-oxidant, had a positive effect on bone healing and improved mechanical strength of the healing bone.


Assuntos
Calo Ósseo/efeitos dos fármacos , Calo Ósseo/diagnóstico por imagem , Força Compressiva/efeitos dos fármacos , Fêmur/efeitos dos fármacos , Fêmur/diagnóstico por imagem , Extrato de Sementes de Uva/administração & dosagem , Animais , Antioxidantes/administração & dosagem , Fenômenos Biomecânicos/fisiologia , Calo Ósseo/fisiologia , Força Compressiva/fisiologia , Suplementos Nutricionais , Fêmur/lesões , Masculino , Ratos , Ratos Wistar
7.
J Orthop Res ; 37(4): 812-820, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30790359

RESUMO

Fractures typically heal via endochondral and intramembranous bone formation, which together form a callus that achieves union and biomechanical recovery. PTHrP, a PTH receptor agonist, plays an important physiological role in fracture healing as an endogenous stimulator of endochondral and intramembranous bone formation. Abaloparatide, a novel systemically-administered osteoanabolic PTH receptor agonist that reduces fracture risk in women with postmenopausal osteoporosis, has 76% homology to PTHrP, suggesting it may have potential to improve fracture healing. To test this hypothesis, ninety-six 12-week-old male rats underwent unilateral internally-stabilized closed mid-diaphyseal femoral fractures and were treated starting the next day with daily s.c. saline (Vehicle) or abaloparatide at 5 or 20 µg/kg/d for 4 or 6 weeks (16 rats/group/time point). Histomorphometry and histology analyses indicated that fracture calluses from the abaloparatide groups exhibited significantly greater total area, higher fluorescence scores indicating more newly-formed bone, and higher fracture bridging scores versus Vehicle controls. Callus bridging score best correlated with callus cartilage score (r = 0.64) and fluorescence score (r = 0.67) at week 4, and callus area correlated with cartilage score (r = 0.60) and fluorescence score (r = 0.89) at Week 6. By micro-CT, calluses from one or both abaloparatide groups had greater bone volume, bone volume fraction, bone mineral content, bone mineral density, and cross-sectional area at both time points versus Vehicle controls. Destructive bending tests indicated greater callus maximum load and stiffness in one or both abaloparatide groups at both time points versus Vehicle controls. These results provide preliminary preclinical evidence for improved fracture healing with systemically-administered abaloparatide. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.


Assuntos
Fraturas do Fêmur/tratamento farmacológico , Consolidação da Fratura/efeitos dos fármacos , Proteína Relacionada ao Hormônio Paratireóideo/uso terapêutico , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Animais , Calo Ósseo/diagnóstico por imagem , Calo Ósseo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Masculino , Proteína Relacionada ao Hormônio Paratireóideo/farmacologia , Ratos Sprague-Dawley , Microtomografia por Raio-X
8.
J Orthop Res ; 37(1): 51-59, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29663560

RESUMO

Fracture healing is a complex process of many coordinated biological pathways. This system can go awry resulting in nonunion, which leads to significant patient morbidity. The Hedgehog (Hh) signaling pathway is upregulated in fracture healing. We hypothesized that the Hh signaling pathway can be pharmacologically modulated to positively affect fracture healing. Diaphyseal femur fractures were created in elderly mice (18 months, C57BL/6 females), which have a blunted and delayed healing response compared to younger mice, and were stabilized with intramedullary pins. To activate the Hh pathway we targeted the receptor Smoothened using an agonist (Hh-Ag1.5 [Hh-Ag]) and compared this to a vehicle control. Expression of Hh target genes were significantly increased in the fracture callus of the agonist group compared to controls, indicating pathway activation. Expression of osteogenic and chondrogenic-related genes was greatly upregulated in fracture callus versus intact femora, although Hh agonist treatment did not consistently enhance this response. Blindly graded, radiographic callus healing scores were significantly higher in the Hh-Ag groups at post operative day (POD) 14, indicating earlier callus bridging. On microCT, Hh-Ag treatment led to greater callus volume (+40%) and bone volume (+25%) at POD21. By day 14, callus vascularity, as assessed by 3D microCT angiography vessel volume, was 85% greater in the Hh-Ag group. Finally, mechanical strength of the calluses in the Hh-Ag groups was significantly greater than in the control groups at POD21. In conclusion, systemic administration of a Hh agonist appears to improve the osseous and vascular healing responses in a mouse fracture healing-impaired model. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.


Assuntos
Consolidação da Fratura/efeitos dos fármacos , Proteínas Hedgehog/agonistas , Fatores Etários , Animais , Calo Ósseo/diagnóstico por imagem , Calo Ósseo/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Feminino , Fraturas do Fêmur/tratamento farmacológico , Expressão Gênica/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Neovascularização Fisiológica/efeitos dos fármacos , Microtomografia por Raio-X
9.
J Orthop Surg Res ; 13(1): 267, 2018 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-30359257

RESUMO

BACKGROUND: Delay in fracture healing or non-union can be devastating complication. Recent studies have reported that teriparatide (TP) demonstrated effectively on callus formation and mechanical strength and zoledronate (ZA) increased the callus size and resistance at the fracture site in rat fracture model. In this study, the effects of combination therapy with low dose TP and ZA on fracture healing was evaluated. METHODS: From 1 week post-operation, TP (5 times a week administration) and ZA (0.1 mg/kg single administration) were administered by dividing the rats into the following five groups: TP 1 µg group {T(1): TP 1 µg/kg}, ZA group (ZA:0.1 mg/kg), TP1 µg+ZA group {T(1)+ZA: TP 1 µg/kg+ZA}, TP 10 µg+ZA group {T(10)+ZA: TP 10 µg/kg + ZA}, and control group (C: administered saline). Rt femurs were excised 7 weeks after the surgery; bone fusions were evaluated with soft X-ray images on a 4-point scale. And the histopathological examination was performed in demineralized and non-demineralized specimens. Furthermore, the Radiographic Union Scale was conducted in all specimens. RESULTS: About the bone fusions rates, C, T(1), ZA, T(1)+ZA, and T(10)+ZA groups demonstrated 20.0%, 55.6%, 70.0%, 70.0%, and 80.0%, respectively, and with 4-point scale, each group was 0.50, 1.56, 2.00, 2.60, and 2.80 points, respectively. The callus volume was significantly increased to 16.66 mm2 and 17.75 mm2 in the T(1)+ZA and T(10)+ZA groups, respectively, while 10.65 mm2 (p < 0.05) in the C group. Furthermore, the callus area in the T(10)+ZA group was also observed to have significantly increased to 78.78%, compared with 54.63% and 44.11% in the C and T(1)+ZA groups, respectively (p < 0.01). Histopathologically, cartilage tissue and immature callus formation were observed at the bone junction in the C group; however, the osseous bridge formation of mature callus was observed in the ZA, T(1)+ZA, and T(10)+ZA groups. CONCLUSION: It is suggested that administration of low dose TP and ZA in combination may lead to the treatment of delayed union of fracture. We hope the combination treatment may become one of new therapeutic strategy.


Assuntos
Conservadores da Densidade Óssea/uso terapêutico , Fraturas do Fêmur/tratamento farmacológico , Consolidação da Fratura/efeitos dos fármacos , Teriparatida/uso terapêutico , Ácido Zoledrônico/uso terapêutico , Animais , Conservadores da Densidade Óssea/administração & dosagem , Conservadores da Densidade Óssea/farmacologia , Calo Ósseo/efeitos dos fármacos , Calo Ósseo/patologia , Modelos Animais de Doenças , Esquema de Medicação , Avaliação Pré-Clínica de Medicamentos/métodos , Quimioterapia Combinada , Fraturas do Fêmur/diagnóstico por imagem , Fraturas do Fêmur/patologia , Fraturas do Fêmur/fisiopatologia , Masculino , Radiografia , Ratos Sprague-Dawley , Teriparatida/administração & dosagem , Teriparatida/farmacologia , Ácido Zoledrônico/administração & dosagem , Ácido Zoledrônico/farmacologia
10.
Cell Mol Biol (Noisy-le-grand) ; 64(7): 92-96, 2018 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-29974852

RESUMO

Arctium lappa L. (Burdock) is an important plant with various pharmacological effects. According to the importance of this plant, optimization of its tissue culture will lead to more investigation and application of it. The aim of this study was to develop protocols for callus induction and shoot regeneration of A.  lappa. In order to optimize of tissue culture in A. lappa, callus induction, indirect regeneration and direct regeneration were carried out in factorial experiment based on Completely Randomized Designs (CRDs). Hypocotyl and cotyledon were cultured on the Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of 2,4-Dichlorophenoxyacetic acid (2,4-D) and 6-Benzylaminopurine (BAP) for callus induction. In indirect regeneration experiment various levels of BAP and α-Naphthaleneacetic acid (NAA) and two types of explants (calli derived from cotyledon and hypocotyl) were investigated. In direct regeneration section, various levels of BAP plus 2 mg/l NAA and different explants (cotyledon, hypocotyl and bud) were compared. In both cotyledon and hypocotyl, the maximum callus induction was observed on a media containing 2 mg/l 2,4-D plus 1 mg/l BAP (100% and 76.19% respectively). The highest percentage of indirect regeneration (65%) was observed at 1 mg/l BAP plus 0.5 mg/l NAA on calli from hypocotyl. The highest percentage of direct regeneration (90.33) was observed in hypocotyl with a lateral bud explant on MS medium supplemented with 0.5 mg/l BAP plus 2 mg/l NAA. In this study, optimization of tissue culture protocol for A. lappa was carried out as a research technique, as well as technique for further exploitation of this plant.


Assuntos
Arctium/fisiologia , Diferenciação Celular/efeitos dos fármacos , Organogênese Vegetal/efeitos dos fármacos , Plantas Medicinais/fisiologia , Regeneração/efeitos dos fármacos , Ácido 2,4-Diclorofenoxiacético/administração & dosagem , Arctium/efeitos dos fármacos , Compostos de Benzil/administração & dosagem , Calo Ósseo/efeitos dos fármacos , Cotilédone/efeitos dos fármacos , Meios de Cultura/farmacologia , Hipocótilo/efeitos dos fármacos , Ácidos Naftalenoacéticos/administração & dosagem , Reguladores de Crescimento de Plantas/farmacologia , Plantas Medicinais/efeitos dos fármacos , Purinas/administração & dosagem , Técnicas de Cultura de Tecidos
11.
J Orthop Res ; 36(3): 930-936, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28767180

RESUMO

Tibial pseudarthrosis associated with Neurofibromatosis type 1 (NF1) is an orthopedic condition with consistently poor clinical outcomes. Using a murine model that features localized double inactivation of the Nf1 gene in an experimental tibial fracture, we tested the effects of recombinant human bone morphogenetic protein-2 (rhBMP-2) and/or the bisphosphonate zoledronic acid (ZA). Tibiae were harvested at 3 weeks for analysis, at which time there was negligible healing in un-treated control fractures (7% union). In contrast, rhBMP-2 and rhBMP-2/ZA groups showed significantly greater union (87% and 93%, p < 0.01 for both). Treatment with rhBMP-2 led to a 12-fold increase in callus bone volume and this was further increased in the rhBMP-2/ZA group. Mechanical testing of the healed rhBMP-2 and rhBMP-2/ZA fractures showed that the latter group had significantly higher mechanical strength and was restored to that of the un-fractured contralateral leg. Co-treatment with rhBMP-2/ZA also reduced fibrous tissue infiltration at the fracture site compared to rhBMP alone (p = 0.068). These data support the future clinical investigation of this combination of anabolic and anti-resorptive agents for the treatment of NF1 pseudarthrosis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:930-936, 2018.


Assuntos
Conservadores da Densidade Óssea/uso terapêutico , Proteína Morfogenética Óssea 2/uso terapêutico , Neurofibromatose 1/complicações , Pseudoartrose/genética , Fator de Crescimento Transformador beta/uso terapêutico , Ácido Zoledrônico/uso terapêutico , Animais , Conservadores da Densidade Óssea/farmacologia , Proteína Morfogenética Óssea 2/farmacologia , Calo Ósseo/efeitos dos fármacos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Quimioterapia Combinada , Feminino , Genes da Neurofibromatose 1 , Camundongos , Pseudoartrose/tratamento farmacológico , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Fator de Crescimento Transformador beta/farmacologia , Ácido Zoledrônico/farmacologia
12.
PLoS One ; 12(10): e0185446, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29020057

RESUMO

Small animal studies have demonstrated significant high-dose recombinant parathyroid hormone1-34 (rPTH1-34) effects on intercalary allograft healing. Towards a human adjuvant therapy to decrease non-unions, we evaluated rPTH1-34 safety and efficacy in a clinically relevant canine femoral allograft model. Adult female mongrel hounds (n = 20) received a 5cm mid-diaphyseal osteotomy reconstructed with a plated allograft, and were randomized to: 1) Placebo (n = 5; daily saline), 2) Continuous rPTH1-34 (n = 7; 5 µg/kg/day s.c. from day 1-55 post-op), or 3) Delayed rPTH1-34 (n = 8; 5 µg/kg/day s.c. from day 14-28 post-op). Safety was assessed by physical behavior and blood calcium monitoring. Cone beam CT (CB-CT) was performed on days 14, 28 and 56 post-op to assess 2D cortical healing, 3D bone volume, and Union Ratio. Biomechanical testing and dynamic histomorphometry were also performed. The high drug dose was poorly tolerated, as most dogs receiving rPTH1-34 had to be given intravenous saline, and one dog died from hypercalcemia. Continuous rPTH1-34 significantly increased 2D healing and callus volumes at 4-weeks versus Placebo, and sustained the significant increase in cortical union at 8-week (p<0.05). These rPTH1-34 effects were confirmed by histomorphometry, revealing significant increases in mineral apposition rates (MAR) on host bone and graft-host junctions (p<0.05). Delayed rPTH1-34 significantly increased callus volume and MAR at 8 weeks (p<0.05). Although no biomechanical differences were observed, as expected for early healing, the results demonstrated that 2D RUST scoring significantly correlated with torsional biomechanics (p<0.01). In conclusion, 8-weeks of intermittent high-dose rPTH1-34 treatment significantly increases callus formation and accelerates bony union of intercalary massive allografts in a clinically relevant canine model, but with serious side-effects from hypercalcemia.


Assuntos
Calo Ósseo/diagnóstico por imagem , Calo Ósseo/patologia , Fêmur/transplante , Teriparatida/administração & dosagem , Teriparatida/uso terapêutico , Cicatrização , Aloenxertos/efeitos dos fármacos , Animais , Fenômenos Biomecânicos/efeitos dos fármacos , Calo Ósseo/efeitos dos fármacos , Tomografia Computadorizada de Feixe Cônico , Modelos Animais de Doenças , Cães , Relação Dose-Resposta a Droga , Fêmur/fisiopatologia , Fêmur/cirurgia , Fluorescência , Minerais/metabolismo , Cuidados Pós-Operatórios , Teriparatida/farmacologia , Cicatrização/efeitos dos fármacos
13.
Osteoporos Int ; 28(10): 3043-3053, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28808745

RESUMO

Ovariectomized (OVX) rats with type 2 diabetes mellitus (T2DM) with femur fracture received vehicle, insulin, or insulin plus parathyroid hormone (PTH) treatment for 2 and 3 weeks. Radiography, histomorphometry, histology, and immunohistochemistry in callus were evaluated. INTRODUCTION: Reports about effects of PTH plus insulin on callus formation of osteoporotic fracture with T2DM were limited. This study was designed to investigate the effects of the combination of PTH and insulin on fracture healing in OVX rats with T2DM. METHODS: Two-month-old female rats were randomly divided into five groups: normal fracture (F), OVX fracture (OF), T2DM + OVX fracture (DOF), insulin-treated (2-4 u/daylight, 4-6 u/night, DOFI), and treated with insulin and PTH (50 µg/kg/day, 5 days/week, DOFIP). A closed mid-shaft fracture was established in the right femurs of all rats after 6 weeks of OVX. Rats were euthanized at 2 and 3 weeks post-fracture according to the time schedule, respectively. RESULTS: The administration of insulin alone or insulin combined with PTH significantly increased mineralized bone volume fraction (BV/TV) and connectivity density (Conn.D) compared with those of the DOF group at 3 weeks post-fracture and also increased cartilaginous callus area ratio in the DOFI and DOFIP groups at 2 weeks and bony callus area ratio in the DOFIP groups at both the 2 and 3 weeks post-fracture. CONCLUSIONS: OVX rats with T2DM exhibited a marked delay in the fracture healing process; insulin treatment ameliorated these effects, and the healing process was enhanced following treatment with a combination of insulin and PTH.


Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Fraturas do Fêmur/tratamento farmacológico , Consolidação da Fratura/efeitos dos fármacos , Hormônio Paratireóideo/uso terapêutico , Animais , Glicemia/metabolismo , Calo Ósseo/diagnóstico por imagem , Calo Ósseo/efeitos dos fármacos , Calo Ósseo/patologia , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos/métodos , Quimioterapia Combinada , Feminino , Fraturas do Fêmur/diagnóstico por imagem , Fraturas do Fêmur/fisiopatologia , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Fraturas por Osteoporose/diagnóstico por imagem , Fraturas por Osteoporose/tratamento farmacológico , Fraturas por Osteoporose/fisiopatologia , Ovariectomia , Hormônio Paratireóideo/farmacologia , Radiografia , Ratos Sprague-Dawley , Microtomografia por Raio-X
14.
Molecules ; 22(1)2017 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-28085103

RESUMO

The genetically transformed hairy root line LRT 7.31 obtained by infecting leaf explants of Lopezia racemosa Cav with the Agrobacterium rhizogenes strain ATCC15834/pTDT, was evaluated to identify the anti-inflammatory and cytotoxic compounds reported previously for the wild plant. After several subcultures of the LRT 7.31 line, the bio-guided fractionation of the dichloromethane-methanol (1:1) extract obtained from dry biomass afforded a fraction that showed important in vivo anti-inflammatory, and in vitro cytotoxic activities. Chemical separation of the active fraction allowed us to identify the triterpenes ursolic (1) and oleanolic (2) acids, and (23R)-2α,3ß,23,28-tetrahydroxy-14,15-dehydrocampesterol (3) as the anti-inflammatory principles of the active fraction. A new molecule 3 was characterized by spectroscopic analysis of its tetraacetate derivative 3a. This compound was not described in previous reports of callus cultures, in vitro germinated seedlings and wild plant extracts of whole L. racemosa plants. The anti-inflammatory and cytotoxic activities displayed by the fraction are associated to the presence of compounds 1-3. The present study reports the obtaining of the transformed hairy roots, the bioguided isolation of the new molecule 3, and its structure characterization.


Assuntos
Anti-Inflamatórios/farmacologia , Proliferação de Células/efeitos dos fármacos , Colesterol/análogos & derivados , Germinação/efeitos dos fármacos , Onagraceae/química , Fitosteróis/farmacologia , Agrobacterium/química , Agrobacterium/genética , Anti-Inflamatórios/química , Calo Ósseo/efeitos dos fármacos , Calo Ósseo/crescimento & desenvolvimento , Colesterol/química , Colesterol/farmacologia , Ácido Oleanólico/química , Ácido Oleanólico/farmacologia , Fitosteróis/química , Raízes de Plantas/química , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plântula/efeitos dos fármacos
15.
Chem Biol Interact ; 261: 11-17, 2017 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-27833010

RESUMO

Postmenopausal osteoporosis is characterized by a reduction in the number of sinusoidal and arterial capillaries in the bone marrow and reduced bone perfusion. Thus, osteogenesis and angiogenesis are coupled in the process of osteoporosis formation and fracture healing. Naringin is the main ingredient of the root Rhizoma Drynariae, a traditional Chinese medicine, and it has potential effects on promoting fracture healing. However, whether naringin stimulates angiogenesis in the process of bone healing is unclear. Here, we show that naringin promotes fracture healing through stimulating angiogenesis by regulating the VEGF/VEGFR-2 signaling pathway in osteoporotic rats.


Assuntos
Flavanonas/farmacologia , Consolidação da Fratura/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Osteoporose/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Densidade Óssea/efeitos dos fármacos , Calo Ósseo/efeitos dos fármacos , Calo Ósseo/patologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Osteócitos/efeitos dos fármacos , Osteócitos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
16.
J Orthop Res ; 34(12): 2106-2115, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-26998841

RESUMO

The process of fracture healing is complex, and poor or incomplete healing remains a significant health problem. Proper fracture healing relies upon resident mesenchymal stem cell (MSC) differentiation into chondrocytes and osteoblasts, which are necessary for callus formation and ossification. Alcohol abuse is a leading contributor to poor fracture healing. Although the mechanism behind this action is unknown, excessive alcohol consumption is known to promote systemic oxidative stress. The family of FoxO transcription factors is activated by oxidative stress, and FoxO activation antagonizes Wnt signaling, which regulates mesenchymal stem cell differentiation. We hypothesize that alcohol exposure increases oxidative stress leading to deficient fracture repair by activating FoxO transcription factors within the fracture callus which disrupts chondrogenesis of mesenchymal stem cells. Our laboratory has developed an experimental model of delayed fracture union in mice using ethanol administration. We have found that ethanol administration significantly decreases external, cartilaginous callus formation, and hallmarks of endochondral ossification, and these changes are concomitant with increases in FoxO expression and markers of activation in fracture callus tissue of these mice. We were able to prevent these alcohol-induced effects with the administration of the antioxidant n-acetyl cysteine (NAC), suggesting that alcohol-induced oxidative stress produces the perturbed endochondral ossification and FoxO expression. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2106-2115, 2016.


Assuntos
Calo Ósseo/efeitos dos fármacos , Depressores do Sistema Nervoso Central/efeitos adversos , Etanol/efeitos adversos , Fatores de Transcrição Forkhead/metabolismo , Consolidação da Fratura/efeitos dos fármacos , Acetilcisteína/farmacologia , Acetilcisteína/uso terapêutico , Animais , Calo Ósseo/metabolismo , Condrogênese/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Sequestradores de Radicais Livres/farmacologia , Sequestradores de Radicais Livres/uso terapêutico , Masculino , Camundongos Endogâmicos C57BL , Distribuição Aleatória
17.
Appl Biochem Biotechnol ; 179(1): 46-58, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26758711

RESUMO

Fagonia indica, a very important anticancer plant, has been less explored for its in vitro potential. This is the first report on thidiazuron (TDZ)-mediated callogenesis and elicitation of commercially important phenolic compounds. Among the five different plant growth regulators tested, TDZ induced comparatively higher fresh biomass, 51.0 g/100 mL and 40.50 g/100 mL for stem and leaf explants, respectively, after 6 weeks of culture time. Maximum total phenolic content (202.8 µg gallic acid equivalent [GAE]/mL for stem-derived callus and 161.3 µg GAE/mL for leaf-derived callus) and total flavonoid content (191.03 µg quercetin equivalent [QE]/mL for stem-derived callus and 164.83 µg QE/mL for leaf-derived callus) were observed in the optimized callus cultures. The high-performance liquid chromatography (HPLC) data indicated higher amounts of commercially important anticancer secondary metabolites such as gallic acid (125.10 ± 5.01 µg/mL), myricetin (32.5 ± 2.05 µg/mL), caffeic acid (12.5 ± 0.52 µg/mL), catechin (9.4 ± 1.2 µg/mL), and apigenin (3.8 ± 0.45 µg/mL). Owing to the greater phenolic content, a better 2-2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity (69.45 % for stem explant and 63.68 % for leaf explant) was observed in optimized calluses. The unusually higher biomass and the enhanced amount of phenolic compounds as a result of lower amounts of TDZ highlight the importance of this multipotent hormone as elicitor in callus cultures of F. indica.


Assuntos
Antioxidantes/química , Fenóis/metabolismo , Extratos Vegetais/química , Zygophyllaceae/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Antioxidantes/farmacologia , Calo Ósseo/efeitos dos fármacos , Calo Ósseo/crescimento & desenvolvimento , Calo Ósseo/metabolismo , Técnicas de Cultura de Células , Flavonoides/química , Fenóis/química , Compostos de Fenilureia/farmacologia , Extratos Vegetais/farmacologia , Folhas de Planta/química , Tiadiazóis/farmacologia , Zygophyllaceae/química , Zygophyllaceae/citologia , Zygophyllaceae/efeitos dos fármacos
18.
Osteoporos Int ; 27(1): 211-8, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26294293

RESUMO

UNLABELLED: This randomized and controlled study evaluated the effect of therapy with strontium ranelate on callus formation in wrist fractures and its incidence in wrist recovery. Radiographic healing, progression of clinical recovery, and callus quality with ultrasound were evaluated. No statistically significant benefit of therapy was found. INTRODUCTION: Fracture prevention is the main goal of any therapy for osteoporosis. Various drugs used in osteoporosis treatment have the theoretical premises to promote fracture healing and osseointegration. In this study, the effect of strontium ranelate on callus formation in wrist fractures was evaluated and whether it could lead to clinically relevant modification of wrist recovery; having strontium ranelate osteoinductive properties, it could be used, if effective, as an adjunct in fracture healing for a faster and functionally better recovery and, at the same time, in starting proper therapy in osteoporotic patients with fragility fractures. METHODS: We considered only patients older than 60 years who had suffered wrist fracture and received nonoperative treatment with manual reduction of the fracture and cast for 35 days. Forty patients were included and randomly assigned to one of two groups: group A [patients treated with calcium (1200 mg/day) and vitamin D (800 IU/day)] and group B [patients treated with calcium (1200 mg/day) and vitamin D (800 IU/day) associated with strontium ranelate 2 g daily]. Radiographic healing was evaluated through the bone callus formation, cortical continuity, and density of the callus. A clinical evaluation using Castaing's criteria was carried out 2 and 3 months following the fracture together with an ultrasound study of callus density and vessels. RESULTS: A parametric analysis of the X-ray data, clinical evaluation, and ultrasonography results showed that there were no statistically significant differences in the two groups (p > 0.05 for all data). CONCLUSION: In analyzing the data obtained, we concluded that strontium ranelate administered in acute phase did not improve nor accelerate wrist fracture healing in our population.


Assuntos
Conservadores da Densidade Óssea/uso terapêutico , Consolidação da Fratura/efeitos dos fármacos , Fraturas por Osteoporose/tratamento farmacológico , Tiofenos/uso terapêutico , Traumatismos do Punho/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Calo Ósseo/diagnóstico por imagem , Calo Ósseo/efeitos dos fármacos , Moldes Cirúrgicos , Quimioterapia Adjuvante , Feminino , Fixação de Fratura/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Fraturas por Osteoporose/diagnóstico por imagem , Radiografia , Resultado do Tratamento , Ultrassonografia , Traumatismos do Punho/diagnóstico por imagem
19.
J Orthop Res ; 34(2): 320-30, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26185108

RESUMO

ACE-011 is a bone anabolic agent generated by fusing the extracellular domain of the Activin Type 2A receptor (ActRIIA) to an IgG-Fc. The orthopedic utility of ACE-011 was investigated using a murine analogue, RAP-011. Initially, a rat closed fracture model was tested using bi-weekly (biw) 10 mg/kg RAP-011. RAP-011 significantly increased callus length and callus bone volume (BV, +43% at 6w, p < 0.01). The polar moment of inertia was calculated to be substantively increased (+80%, p < 0.01), however mechanical bending tests showed a more modest increase in maximum load to failure (+24%, p < 0.05). Histology indicated enhanced appositional bone growth, but it was hypothesized that reduced remodeling, evidenced by decreased serum CTX (-16% at 6w, p < 0.01), could be compromising bone quality in the callus. A second closed fracture study was performed to examine lower "pulse" [RAP-011(p)] and "sustained" [RAP-011(s)] regimens of biw 0.6mg/kg × 2, 0.35mg/kg × 3 and 0.18mg/kg × 2, 0.1mg/kg × 7 respectively, compared with PTH(1-34) (25 µg/kg/d) and vehicle controls. RAP-011 treatments gave modest increases in callus length and callus BV at 6w (p < 0.01), but did not achieve an increase in maximum load over vehicle. In summary, RAP-011 is effective in promoting bone formation during repair, but optimizing callus bone quality will require further investigation.


Assuntos
Fraturas Ósseas/tratamento farmacológico , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Fenômenos Biomecânicos , Calo Ósseo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Masculino , Ratos Wistar , Proteínas Recombinantes de Fusão/farmacologia
20.
J Orthop Surg Res ; 10: 88, 2015 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-26037517

RESUMO

BACKGROUND: Changes over time in the callus during intermittent administration of parathyroid hormone (PTH) were studied in rabbit distraction osteogenesis models. METHOD: Models of distraction osteogenesis in Japanese white rabbits were created, and distraction osteogenesis (total length: 10.5 mm) was performed for 2 weeks. Simultaneously with the start of distraction, 30 rabbits received 4 weeks of subcutaneous administration of 30 µg/kg of PTH(1-34), teriparatide, (P-group: n = 15) or saline (N-group: n = 15) every other day. The tibias of five rabbits were dissected at 6, 8, and 10 weeks after surgery to perform bone mineral density (BMD), peripheral quantitative computed tomography (pQCT), and mechanical testing. RESULTS: The mean BMD had no significant differences over time at 6, 8, and 10 weeks after surgery between the P-group and the N-group. On pQCT, the P-group had significant increases in total bone cross-sectional area of the callus compared to the N-group at 8 and 10 weeks after surgery. On mechanical testing, the P-group's absorption energy had not changed at 6 weeks after surgery compared to the N-group, but it had significantly increased at 8 weeks. At 10 weeks after surgery, the N-group's absorption energy rapidly increased, and the difference between the two groups disappeared. CONCLUSION: The intermittent administration of PTH(1-34), teriparatide, for 4 weeks every other day from the start of distraction had the potential to shorten the callus maturation period in the rabbit distraction osteogenesis models.


Assuntos
Conservadores da Densidade Óssea/administração & dosagem , Calo Ósseo/efeitos dos fármacos , Osteogênese por Distração , Teriparatida/administração & dosagem , Animais , Densidade Óssea/efeitos dos fármacos , Calo Ósseo/diagnóstico por imagem , Avaliação Pré-Clínica de Medicamentos , Modelos Animais , Coelhos , Tomografia Computadorizada por Raios X , Suporte de Carga
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