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1.
Cell Tissue Res ; 387(2): 275-285, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34820705

RESUMO

Isosteviol has been indicated as a cardiomyocyte protector. However, the underlying mechanism remains unclear. Thus, we sought to confirm the protective effect of isosteviol after myocardial infarction in a model of permanent coronary artery occlusion and investigate the potential proangiogenic activity in vitro and in vivo. A 4-week permanent coronary artery occlusion rat model was generated, and the protective effect of isosteviol was evaluated by echocardiographic imaging and hemodynamics assays. The coronary capillary density was tested by immunochemistry and micro-computed tomography (µCT) imaging. The effect of isosteviol on endothelial cells was determined in human umbilical vein endothelial cells (HUVECs) in vitro and Tg (kdrl: EGFP) zebrafish in vivo. We also examined the expression of related transcription factors by real-time polymerase chain reaction (RT-qPCR). Isosteviol increased ejection fraction (EF), fractional shortening (FS), cardiac systolic index (CI), maximum rate of increase of left ventricular pressure (Max dp/dt), and left ventricular systolic pressure (LVSP) by 32%, 40%, 25%, 26%, and 10%, respectively, in permanent coronary artery occlusion rats. Interestingly, it also promoted coronary capillary density by 2.5-fold. In addition, isosteviol promoted the proliferation and branching of HUVECs in vitro. It also rescued intersegmental vessel (ISV) development and improved endothelial cell proliferation by approximately fivefold (4-6) in zebrafish embryos in vivo. Isosteviol also upregulated the expression of hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor A (VEGFA) in zebrafish by fourfold and 3.5-fold, respectively. Our findings suggest that isosteviol is a proangiogenic agent and that this activity is related to its protective effects against myocardial ischemia. After using the permanent coronary artery occlusion model, we demonstrated that isosteviol promotes angiogenesis directly and increases capillary density in myocardial ischemia rats. Isosteviol promotes angiogenesis in zebrafish in vivo and increases vascular endothelial cell proliferation in HUVECs and zebrafish. The angiogenesis activity of isosteviol may be correlated with VEGFA and HIF-1α signaling.


Assuntos
Infarto do Miocárdio , Fator A de Crescimento do Endotélio Vascular , Animais , Diterpenos do Tipo Caurano , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neovascularização Fisiológica , Ratos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Microtomografia por Raio-X , Peixe-Zebra/metabolismo
2.
Drug Des Devel Ther ; 16: 2949-2965, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36090955

RESUMO

Purpose: To investigate the effects of sinomenine on orthodontic tooth movement and root resorption in rats, as well as the effect of sinomenine on the osteogenesis of periodontal ligament stem cells (PDLSCs). Methods: Fifty-four male Wistar rats were randomly divided into 3 groups: control group, 20 mg/kg sinomenine group and 40 mg/kg sinomenine group. Fifty-gram orthodontic force was applied to all groups. Each group was injected intraperitoneally with corresponding concentration of sinomenine every day. After 14 days, all rats were sacrificed. Micro-computed tomography (micro-CT) scan was used to analyze tooth movement, root resorption and alveolar bone changes. The effect on periodontal tissue was analyzed by Masson, tartrate-resistant acid phosphatase (TRAP) and immunohistochemical staining. In vitro, PDLSCs were extracted and identified. The effect of sinomenine on proliferation was determined by cell-counting kit-8. The effect of sinomenine on osteogenesis was investigated by alkaline phosphatase (ALP) activity and alizarin red staining. qPCR and Western blotting were performed to explore the effects of sinomenine on the expression levels of ALP, runt-related transcription factor 2 (RUNX2), receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG). Results: The tooth movement and root resorption of sinomenine groups were reduced. Sinomenine decreased trabecular spacing on compression side and increased alveolar bone volume and trabecular thickness on tension side. TRAP-positive cells in sinomenine groups decreased significantly. The expressions of TNF-α and RANKL were decreased, while the expressions of OPG, RUNX2 and osteocalcin were up-regulated. In vitro, 0.1 M and 0.5 M sinomenine enhanced ALP activity, mineral deposition and the expression of ALP, RUNX2 and OPG, and reduced the expression of RANKL. Conclusion: Sinomenine could inhibit tooth movement, reduce root resorption, and exert a positive effect on bone formation in rats. Moreover, sinomenine promoted the osteogenesis of PDLSCs.


Assuntos
Reabsorção da Raiz , Animais , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Masculino , Morfinanos , Osteogênese , Ligamento Periodontal/metabolismo , Ratos , Ratos Wistar , Reabsorção da Raiz/tratamento farmacológico , Células-Tronco/metabolismo , Técnicas de Movimentação Dentária , Microtomografia por Raio-X
3.
Biomed Res Int ; 2022: 7532434, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36093403

RESUMO

The knee osteoarthritis is a common joint disease that causes pain and inconvenience. Clinically, patients with knee osteoarthritis often have response points on the gastrocnemius. Gastrocnemius plays an essential role in stabilizing joints and changing gait and pace, which also has a close relationship with the knee joint. The objective of this study is to determine changes in the tibiofemoral joint after medial and lateral gastrocnemius injury. Rabbits were divided into a medial gastrocnemius injury group, a lateral gastrocnemius injury group, and a control group with two intervals: 6 and 8 weeks after modeling of the semisevered gastrocnemius. The gastrocnemius was weighed and sectioned for histology. The joint space and subchondral bone were observed using X-ray and microcomputed tomography. The cartilage was observed histologically using Safranin O fast green and Masson and immunohistochemically using antibodies to collagen type II, matrix metalloproteinase 13, and integrin beta1. Results showed muscle fiber atrophy, and fibrotic changes occurred after gastrocnemius semidissociation. After gastrocnemius injury, the femoral condyle of the tibiofemoral joint produced abnormal sclerosis and bone degeneration. The pathological changes of cartilage included disordered or reduced cell alignment, cartilage matrix loss, and collagen loss due to decreased collagen type II and increased matrix metalloproteinase 13 activity. The increase of integrin beta1 in the injured group may be related to mechanical conduction process. The results suggest that gastrocnemius injury is an essential factor in tibiofemoral arthritis.


Assuntos
Doenças das Cartilagens , Cartilagem Articular , Osteoartrite do Joelho , Animais , Doenças das Cartilagens/patologia , Cartilagem Articular/patologia , Colágeno Tipo II , Integrina beta1 , Articulação do Joelho/patologia , Metaloproteinase 13 da Matriz , Músculo Esquelético/patologia , Osteoartrite do Joelho/patologia , Coelhos , Microtomografia por Raio-X
4.
Sci Rep ; 12(1): 15544, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-36109611

RESUMO

Although several studies have analyzed inter-individual differences in the femoral neck cortical microstructure, intra-individual variations have not been comprehensively evaluated. By using microCT, we mapped cortical pore volume fraction (Ct.Po) and thickness (Ct.Th) along the superolateral femoral neck in 14 older women (age: 77.1 ± 9.8 years) to identify subregions and segments with high porosity and/or low thickness-potential "critical" spots where a fracture could start. We showed that Ct.Po and Ct.Th significantly differed between basicervical, midcervical, and subcapital subregions of the femoral neck (p < 0.001), where the subcapital subregion showed the lowest mean Ct.Th and the highest mean Ct.Po. These cortical parameters also varied substantially with age and with the location of the analyzed microsegments along the individual's neck (p < 0.001), showing multiple microsegments with high porosity and/or low thickness. Although the highest ratio of these microsegments was found in the subcapital subregion, they were also present at other examined subregions, which may provide an anatomical basis for explaining the fracture initiation at various sites of the superolateral neck. Given that fractures likely start at structurally and mechanically weaker spots, intra-individual variability in Ct.Po and Ct.Th should be considered and the average values for the entire femoral neck should be interpreted with caution.


Assuntos
Colo do Fêmur , Fraturas Ósseas , Idoso , Idoso de 80 Anos ou mais , Feminino , Fêmur , Colo do Fêmur/diagnóstico por imagem , Humanos , Porosidade , Microtomografia por Raio-X
5.
Sci Rep ; 12(1): 15555, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36114343

RESUMO

A targeted injury to the mouse intervertebral disc (IVD) is often used to recapitulate the degenerative cascade of the human pathology. Since injuries can vary in magnitude and localization, it is critical to examine the effects of different injuries on IVD degeneration. We thus evaluated the degenerative progression resulting from either a partial- or full-width injury to the mouse lumbar IVD using contrast-enhanced micro-computed tomography and histological analyses. A lateral-retroperitoneal surgical approach was used to access the lumbar IVD, and the injuries to the IVD were produced by either incising one side of the annulus fibrosus or puncturing both sides of the annulus fibrosus. Female C57BL/6J mice of 3-4 months age were used in this study. They were divided into three groups to undergo partial-width, full-width, or sham injuries. The L5/6 and L6/S1 lumbar IVDs were surgically exposed, and then the L6/S1 IVDs were injured using either a surgical scalpel (partial-width) or a 33G needle (full-width), with the L5/6 serving as an internal control. These animals recovered and then euthanized at either 2-, 4-, or 8-weeks after surgery for evaluation. The IVDs were assessed for degeneration using contrast-enhanced microCT (CEµCT) and histological analysis. The high-resolution 3D CEµCT evaluation of the IVD confirmed that the respective injuries were localized within one side of the annulus fibrosus or spanned the full width of the IVD. The full-width injury caused significant deteriorations in the nucleus pulposus, annulus fibrous and at the interfaces after 2 weeks, which was sustained through the 8 weeks, while the partial width injury caused localized disruptions that remained limited to the annulus fibrosus. The use of CEµCT revealed distinct IVD degeneration profiles resulting from partial- and full-width injuries. The partial width injury may serve as an alternative model for IVD degeneration resulting from localized annulus fibrosus injuries.


Assuntos
Anel Fibroso , Degeneração do Disco Intervertebral , Disco Intervertebral , Animais , Anel Fibroso/diagnóstico por imagem , Anel Fibroso/patologia , Feminino , Humanos , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/diagnóstico por imagem , Degeneração do Disco Intervertebral/patologia , Camundongos , Camundongos Endogâmicos C57BL , Punção Espinal , Microtomografia por Raio-X
6.
Biomed Eng Online ; 21(1): 68, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36114576

RESUMO

BACKGROUND: Medial compartment knee osteoarthritis (KOA) accounts for most KOA cases, and increased trabecular bone volume fraction (BV/TV) is one of the pathological changes in the tibial plateau of KOA. How BV/TV changes before and after the menopause and its effects on medial compartment KOA are yet to be clarified. METHODS: Twenty femurs from twenty 12-week-old rats were included. The operated group underwent ovariectomy (to represent the osteoporosis condition), called the O group, and the non-operated group was the normal control, called the N group. Micro-CT scans of the femoral condyles were acquired 12 weeks after the surgery, and the volume of interest (VOI) of medial-, inter-, and lateral-condyle trabeculae were three-dimensional (3D) printed for uniaxial compression mechanical test and simulated by the finite element (FE) method. RESULTS: The results demonstrated that the O group indicated poorer trabecular architecture than the N group in three parts of the femoral condyle, especially in the intercondyle. Within the group, the BV/TV, trabecular thickness (Tb.Th), and trabecular number (Tb.N) ratios between the medial and lateral condyles were greater than 1 in both N and O groups. The medial condyle trabeculae's mechanical properties were higher than those of the lateral condyle, and this superiority appears to be broadened under osteoporotic conditions. FE modelling well reproduced these mechanical differentiations. CONCLUSIONS: According to Wolff's law, the higher BV/TV and mechanical properties of the medial femoral condyle may be due to inherent imbalanced loading on the knee component. Alterations in BV/TV and their corresponding mechanical properties may accompany KOA.


Assuntos
Osteoartrite do Joelho , Animais , Feminino , Análise de Elementos Finitos , Osteoartrite do Joelho/diagnóstico por imagem , Impressão Tridimensional , Ratos , Estresse Fisiológico , Microtomografia por Raio-X
7.
Front Endocrinol (Lausanne) ; 13: 910901, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36046782

RESUMO

Unloading associated with spaceflight results in bone loss and increased fracture risk. Bone morphogenetic protein 2 (BMP2) is known to enhance bone formation, in part, through molecular pathways associated with mechanical loading; however, the effects of BMP2 during spaceflight remain unclear. Here, we investigated the systemic effects of BMP2 on mice sustaining a femoral fracture followed by housing in spaceflight (International Space Station or ISS) or on Earth. We hypothesized that in spaceflight, the systemic effects of BMP2 on weight-bearing bones would be blunted compared to that observed on Earth. Nine-week-old male mice were divided into four groups: 1) Saline+Earth; 2) BMP+Earth; 3) Saline+ISS; and 4) BMP+ISS (n = 10 mice/group, but only n = 5 mice/group were reserved for micro-computed tomography analyses). All mice underwent femoral defect surgery and were followed for approximately 4 weeks. We found a significant reduction in trabecular separation within the lumbar vertebrae after administering BMP2 at the fracture site of mice housed on Earth. In contrast, BMP2 treatment led to a significant increase in trabecular separation concomitant with a reduction in trabecular number within spaceflown tibiae. Although these and other lines of evidence support our hypothesis, the small sample size associated with rodent spaceflight studies limits interpretations. That said, it appears that a locally applied single dose of BMP2 at the femoral fracture site can have a systemic impact on distant bones, affecting bone quantity in several skeletal sites. Moreover, our results suggest that BMP2 treatment works through a pathway involving mechanical loading in which the best outcomes during its treatment on Earth occurred in the weight-bearing bones and in spaceflight occurred in bones subjected to higher muscle contraction.


Assuntos
Fraturas do Fêmur , Voo Espacial , Animais , Proteína Morfogenética Óssea 2 , Osso e Ossos , Fraturas do Fêmur/diagnóstico por imagem , Fraturas do Fêmur/etiologia , Masculino , Camundongos , Microtomografia por Raio-X
8.
Front Endocrinol (Lausanne) ; 13: 909317, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36060973

RESUMO

Objective: To investigate the effects of vibration therapy on fracture healing in diabetic and non-diabetic rats. Methods: 148 rats underwent fracture surgery and were assigned to four groups: (1) SHAM: weight-matched non-diabetic rats, (2) SHAM+VT: non-diabetic rats treated with vibration therapy (VT), (3) DM: diabetic rats, and (4) DM+VT: diabetic rats treated with VT. Thirty days after diabetes induction with streptozotocin, animals underwent bone fracture, followed by surgical stabilization. Three days after bone fracture, rats began VT. Bone healing was assessed on days 14 and 28 post-fracture by serum bone marker analysis, and femurs collected for dual-energy X-ray absorptiometry, micro-computed tomography, histology, and gene expression. Results: Our results are based on 88 animals. Diabetes led to a dramatic impairment of bone healing as demonstrated by a 17% reduction in bone mineral density and decreases in formation-related microstructural parameters compared to non-diabetic control rats (81% reduction in bone callus volume, 69% reduction in woven bone fraction, 39% reduction in trabecular thickness, and 45% in trabecular number). These changes were accompanied by a significant decrease in the expression of osteoblast-related genes (Runx2, Col1a1, Osx), as well as a 92% reduction in serum insulin-like growth factor I (IGF-1) levels. On the other hand, resorption-related parameters were increased in diabetic rats, including a 20% increase in the callus porosity, a 33% increase in trabecular separation, and a 318% increase in serum C terminal telopeptide of type 1 collagen levels. VT augmented osteogenic and chondrogenic cell proliferation at the fracture callus in diabetic rats; increased circulating IGF-1 by 668%, callus volume by 52%, callus bone mineral content by 90%, and callus area by 72%; and was associated with a 19% reduction in circulating receptor activator of nuclear factor kappa beta ligand (RANK-L). Conclusions: Diabetes had detrimental effects on bone healing. Vibration therapy was effective at counteracting the significant disruption in bone repair induced by diabetes, but did not improve fracture healing in non-diabetic control rats. The mechanical stimulus not only improved bone callus quality and quantity, but also partially restored the serum levels of IGF-1 and RANK-L, inducing bone formation and mineralization, thus creating conditions for adequate fracture repair in diabetic rats.


Assuntos
Diabetes Mellitus , Fraturas Ósseas , Animais , Calo Ósseo/metabolismo , Calo Ósseo/patologia , Diabetes Mellitus/patologia , Consolidação da Fratura , Fraturas Ósseas/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Ratos , Vibração/uso terapêutico , Microtomografia por Raio-X
9.
In Vivo ; 36(5): 2126-2133, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36099088

RESUMO

BACKGROUND/AIM: Idiopathic condylar resorption (ICR) is a morphological change of the condylar head that occurs following orthodontic treatment or orthognathic surgery. This complication is serious, as it can cause relapse after mandible treatment. The aim of this experimental study was to evaluate the mechanism of influence of condylar resorption on compressive mechanical stress in temporomandibular joint following a change in occlusal position by mandible advancement. MATERIALS AND METHODS: An osteotomy procedure at the midline of mandible was performed in 15 rabbits, with the left side moved forward by 3.5 mm. Advancement of the left side of the mandible resulted in compressive mechanical stress on condylar head on the left side. Samples were subjected to micro-computed tomography, histological staining and immunohistochemistry. RESULTS: The area and depth of anterior condylar resorption at two weeks were significantly different as compared to those at one week (p<0.05). TRAP staining confirmed the significantly largest number of TRAP-positive cells after two weeks (p=0.02), compared to one week. MMP-3 and MMP-13 immunostaining of the anterior condylar head at two weeks revealed high levels of both proteins from the surface to the deep layer of cartilage. CONCLUSION: Compressive mechanical stress following mandible advancement results in load on the anterior surface of the condylar head, which leads to bone resorption there, and induces MMP-3 and MMP-13 related to degradation of condylar head cartilage.


Assuntos
Côndilo Mandibular , Metaloproteinase 3 da Matriz , Animais , Côndilo Mandibular/patologia , Côndilo Mandibular/cirurgia , Metaloproteinase 13 da Matriz , Coelhos , Estresse Mecânico , Microtomografia por Raio-X
10.
Artigo em Inglês | MEDLINE | ID: mdl-36078295

RESUMO

Soils are dynamic and complex systems in their natural state, which are subjected to profound changes due to management. Additionally, agricultural soils are continuously exposed to wetting and drying (W-D) cycles, which can cause modifications in the complexity of their pores. Thus, we explore how successive W-D cycles can affect the pore network of an Oxisol under contrasting managements (conventional tillage-CT, minimum tillage-MT, no tillage-NT, and secondary forest-F). The complexity of the soil pore architecture was evaluated using a 3D multifractal approach combined with lacunarity, Shannon's entropy, and pore geometric parameters. Our results showed that the multifractal approach effectively identified and quantified the changes produced in the soil pore architecture by the W-D cycles. The lacunarity curves revealed important aspects of the modifications generated by these cycles. Samples under F, NT, and MT suffered the most significant changes. Pore connectivity and tortuosity were largely affected by the cycles in F and NT. Our findings demonstrated that the 3D geometric parameters and normalized Shannon's entropy are complementary types of analysis. According to the adopted management, they allowed us to separate the soil into two groups according to their similarities (F and NT; CT and MT).


Assuntos
Agricultura , Solo , Agricultura/métodos , Microtomografia por Raio-X
11.
Annu Int Conf IEEE Eng Med Biol Soc ; 2022: 3830-3833, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-36086069

RESUMO

The healing of bone fractures is a complex and well-orchestrated physiological process, but normal healing is compromised when the fracture is large. These large non-union fractures often require a template with surgical intervention for healing. The standard treatment, autografting, has drawbacks such as donor site pain and limited availability. Biodegradable scaffolds developed using biomaterials such as bioactive glass are a potential solution. Investigation of bone ingrowth into biodegradable scaffolds is an important aspect of their development. Micro-CT (µ-CT) imaging is widely used to evaluate and quantify tissue ingrowth into scaffolds in 3D. Existing segmentation techniques have low accuracy in differentiating bone and scaffold, and need improvements to accurately quantify the bone in-growth into the scaffold using µ-CT scans. This study proposes a novel 3-stage pipeline for better outcome. The first stage of the pipeline is based on a convolutional neural network for the segmentation of the scaffold, bone, and pores from µ-CT images to investigate bone ingrowth. A 3D rigid image registration procedure was employed in the next stage to extract the volume of interest (VOI) for the analysis. In the final stage, algorithms were developed to quantitatively analyze bone ingrowth and scaffold degradation. The best model for segmentation produced a dice similarity coefficient score of 90.1, intersection over union score of 83.9, and pixel accuracy of 93.1 for unseen test data.


Assuntos
Osso e Ossos , Semântica , Materiais Biocompatíveis , Osso e Ossos/diagnóstico por imagem , Cicatrização , Microtomografia por Raio-X/métodos
12.
Drug Des Devel Ther ; 16: 2885-2900, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36060929

RESUMO

Purpose: Puerarin (C21H20O10) is a phytoestrogen that possesses various pharmacological effect, and several researches have revealed the relationship between puerarin and bone metabolism. This study was aimed to evaluate the potential influence of puerarin on the proliferation and osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (BMSCs) as well as on new bone formation following rapid maxillary expansion (RME) model in rats. Methods: Rat BMSCs were adopted, and the cell proliferation was detected by cell-counting kit-8 (CCK-8) assay in vitro experiments. Alkaline phosphatase (ALP) activity and alizarin red staining were analyzed quantitatively to show extracellular matrix mineralization. The mRNA and protein expression levels were used to detect osteogenic differentiation of BMSCs. In vivo bone regeneration was analyzed in a rat RME model. Eighteen 6-week-old male Wistar rats were divided into 3 groups: group 1 without any treatment, group 2 received RME and saline solution (15mg/kg), group 3 received RME and puerarin solution (15mg/kg). After 2 weeks, micro-computed tomography (Micro-CT), hematoxylin and eosin (HE) staining, and Masson staining were used to detect the new bone formation and morphological changes. Besides, ALP and bone morphogenetic protein 2 (BMP2) expression levels in mid-palatal suture were evaluated by immunohistochemical staining. Results: The results showed that puerarin upregulates cell proliferation dose-dependently. ALP activity and mineralized matrix generation were clearly enhanced at certain specific concentrations (10-5 and 10-6 mol/L); the expression levels of the osteoblast-related genes and proteins were increased. The measurement of micro-CT imaging revealed that puerarin significantly promoted new bone formation. Concomitantly, the histological examinations showed that puerarin solution enhanced osteogenesis in mid-palatal suture. Conclusion: Those works indicated that puerarin regulates osteogenesis in vitro and exerts a beneficial impact on bone regeneration in vivo, revealing that puerarin treatment may become one of the potential keys for improving the stability and preventing relapse of RME.


Assuntos
Células da Medula Óssea , Osteogênese , Animais , Isoflavonas , Masculino , Ratos , Ratos Wistar , Microtomografia por Raio-X
13.
Stem Cell Res Ther ; 13(1): 456, 2022 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-36064451

RESUMO

BACKGROUND: The available therapeutic options for large bone defects remain extremely limited, requiring new strategies to accelerate bone healing. Genetically modified bone mesenchymal stem cells (BMSCs) with enhanced osteogenic capacity are recognised as one of the most promising treatments for bone defects. METHODS: We performed differential expression analysis of miRNAs between human BMSCs (hBMSCs) and human dental pulp stem cells (hDPSCs) to identify osteogenic differentiation-related microRNAs (miRNAs). Furthermore, we identified shared osteogenic differentiation-related miRNAs and constructed an miRNA-transcription network. The Forkhead box protein A1 (FOXA1) knockdown strategy with a lentiviral vector was used to explore the role of FOXA1 in the osteogenic differentiation of MSCs. Cell Counting Kit-8 was used to determine the effect of the knockdown of FOXA1 on hBMSC proliferation; real-time quantitative reverse transcription PCR (qRT-PCR) and western blotting were used to investigate target genes and proteins; and alkaline phosphatase (ALP) staining and Alizarin Red staining (ARS) were used to assess ALP activity and mineral deposition, respectively. Finally, a mouse model of femoral defects was established in vivo, and histological evaluation and radiographic analysis were performed to verify the therapeutic effects of FOXA1 knockdown on bone healing. RESULTS: We identified 22 shared and differentially expressed miRNAs between hDPSC and hBMSC, 19 of which were downregulated in osteogenically induced samples. The miRNA-transcription factor interaction network showed that FOXA1 is the most significant and novel osteogenic differentiation biomarker among more than 300 transcription factors that is directly targeted by 12 miRNAs. FOXA1 knockdown significantly promoted hBMSC osteo-specific genes and increased mineral deposits in vitro. In addition, p-ERK1/2 levels were upregulated by FOXA1 silencing. Moreover, the increased osteogenic differentiation of FOXA1 knockdown hBMSCs was partially rescued by the addition of ERK1/2 signalling inhibitors. In a mouse model of femoral defects, a sheet of FOXA1-silencing BMSCs improved bone healing, as detected by microcomputed tomography and histological evaluation. CONCLUSION: These findings collectively demonstrate that FOXA1 silencing promotes the osteogenic differentiation of BMSCs via the ERK1/2 signalling pathway, and silencing FOXA1 in vivo effectively promotes bone healing, suggesting that FOXA1 may be a novel target for bone healing.


Assuntos
Fator 3-alfa Nuclear de Hepatócito , Células-Tronco Mesenquimais , MicroRNAs , Osteogênese , Animais , Células da Medula Óssea , Diferenciação Celular/genética , Células Cultivadas , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/genética , Células-Tronco Mesenquimais/metabolismo , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Osteogênese/genética , Osteogênese/fisiologia , Microtomografia por Raio-X
14.
Sci Rep ; 12(1): 15228, 2022 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-36075938

RESUMO

Mayflies (Ephemeroptera) are among the oldest pterygote insects, with the earliest fossils dating back to the Late Carboniferous. Within mayflies, Leptophlebiidae are a highly diverse and widespread group, with approximately 140 genera and 640 species. Whereas taxonomy, systematics, and phylogeny of extant Leptophlebiidae are in the focus of extensive studies, little is known about leptophlebiid fossil taxa. Because fossil remains of Ephemeroptera in sedimentary rocks are relatively rare, inclusions of mayflies in amber are a unique source of information on their evolution and diversity in the past. Leptophlebiidae found in Cenozoic resins mostly belong to the subfamilies Leptophlebiinae (in Eocene Baltic amber) and Atalophlebiinae (in Miocene Dominican and Mexican ambers). In the present contribution, we confirm the first finding of the genus Calliarcys from Eocene Baltic amber by using Micro-CT, which allowed confirming its generic placement by visualizing diagnostic key characters otherwise hidden by a cloud of turbidity. Additionally, we present first molecular data on the extant species Calliarcys humilis Eaton, 1881 from the Iberian Peninsula and the barcode gap analysis for Leptophlebiinae and Habrophlebiinae.


Assuntos
Âmbar , Ephemeroptera , Animais , Código de Barras de DNA Taxonômico , Fósseis , Insetos , Microtomografia por Raio-X
15.
Rev Sci Instrum ; 93(8): 083701, 2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-36050103

RESUMO

We describe our miniature laser powder bed fusion (L-PBF) system for in situ synchrotron x-ray micro-computed tomography (XCT) at the European Synchrotron Radiation Facility. This replicator was designed to extend the characterization of L-PBF to 3D. This instrument fills in a technical gap because the existing replicators were mostly designed to shed light on the dynamic mechanisms involved in molten pool formation but, therefore, suffered from a lack of 3D information. Technical details regarding the setup and beamline integration are given. Experimental validations via post-mortem XCT scans and in situ scans acquired during experiments conducted at the BM05 beamline of the European Synchrotron Radiation Facility are provided. Based on a few illustrative examples, we show that such a replicator opens the path to collect key 3D information that to date could not be available. Our miniature instrument complements the other replicators developed in the world by other research groups that enable operando x-ray imaging (radiography) and operando x-ray diffraction.


Assuntos
Síncrotrons , Pós , Difração de Raios X , Microtomografia por Raio-X , Raios X
16.
Sci Rep ; 12(1): 14175, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36050485

RESUMO

While it is well-established that bone responds dynamically to mechanical loading, the effects of mild traumatic brain injury (mTBI) on cranial bone composition are unclear. We hypothesized that repeated mTBI (rmTBI) would change the microstructure of cranial bones, without gross skull fractures. To address this, young adult female Piebald Viral Glaxo rats received sham, 1×, 2× or 3× closed-head mTBIs delivered at 24 h intervals, using a weight-drop device custom-built for reproducible impact. Skull bones were collected at 2 or 10 weeks after the final injury/sham procedure, imaged by micro computed tomography and analyzed at predetermined regions of interest. In the interparietal bone, proximal to the injury site, modest increases in bone thickness were observed at 2 weeks, particularly following 2× and 3× mTBI. By 10 weeks, 2× mTBI induced a robust increase in the volume and thickness of the interparietal bone, alongside a corresponding decrease in the volume of marrow cavities in the diploë region. In contrast, neither parietal nor frontal skull samples were affected by rmTBI. Our findings demonstrate time- and location-dependent effects of rmTBI on cranial bone structure, highlighting a need to consider microstructural alterations to cranial bone when assessing the consequences of rmTBI.


Assuntos
Concussão Encefálica , Lesões Encefálicas Traumáticas , Animais , Concussão Encefálica/diagnóstico por imagem , Modelos Animais de Doenças , Feminino , Ratos , Crânio/diagnóstico por imagem , Tempo , Microtomografia por Raio-X
17.
Tissue Eng Part C Methods ; 28(9): 489-497, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35959744

RESUMO

Establishing a suitable animal model of growth plate injury is necessary to evaluate the effect of tissue engineering scaffolds on repairing the injured growth plate. However, the currently used animal models have limitations. Therefore in this study, we reported and evaluated a new modeling method termed the longitudinal disruption method, which is to make a longitudinal defect in the region of growth plate. To compare this new method with the traditional transverse disruption method, we constructed the models by both methods, respectively. To observe whether bone bridges were formed, histological sections were analyzed by hematoxylin-eosin (HE) and Masson staining at 3 weeks after modeling. The HE and Masson staining results showed the formation of bone bridges in both groups, implying that the two methods successfully injured the growth plate. However, it was unclear whether the exact injury to growth plate caused by both methods was consistent. Therefore, to evaluate the accuracy and precision of modeling method, the X-ray and micro-computed tomography (CT) were performed immediately after modeling. The percentages of accurate defect position in the longitudinal and transverse modeling groups were 88.89% and 55.56%, respectively. The micro-CT results revealed irregularly shaped defect cross sections in the transverse modeling group, whereas the defects in the longitudinal modeling group had regular shapes. The mean defect areas were 10.06 ± 0.86 and 12.30 ± 2.13 mm2 in the longitudinal and transverse modeling groups, respectively, while the difference between the actual area and the expected area were -1.94 ± 0.86 and -7.70 ± 2.13 mm2, respectively, showing the high precision of this new method. Altogether, we successfully demonstrated a new method for establishing a rabbit model of growth plate injury, which provides a simple and rapid modeling process, good modeling effect, high modeling accuracy, and convenient scaffold implantation. The new method provides an effective animal model for tissue engineering research on the repair and regeneration of injured growth plate. Impact Statement In recent years, an increasing number of studies have used tissue engineering scaffolds in the repair and regeneration of growth plate. However, the currently used animal models have certain limitations in the study of tissue engineering scaffold for growth plate. In this study, a new method is presented to establish a rabbit model of growth plate injury. This method is characterized by simple and rapid modeling process, good modeling effect, high modeling accuracy, and convenient scaffold implantation, which is suitable for the study of the repair effects of tissue engineering scaffolds. Altogether, this method provides an effective animal model for tissue engineering research on growth plate and facilitates the development of tissue engineering research on the repair and regeneration of injured growth plate.


Assuntos
Fraturas Salter-Harris , Engenharia Tecidual , Animais , Regeneração Óssea , Amarelo de Eosina-(YS) , Hematoxilina , Osteogênese , Coelhos , Engenharia Tecidual/métodos , Tecidos Suporte , Microtomografia por Raio-X
18.
J Periodontal Res ; 57(5): 1056-1069, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35989621

RESUMO

BACKGROUND AND OBJECTIVES: The potential role of the transcription factor Differentiated embryo-chondrocyte 2 (Dec2) in the progression of inflammatory diseases such as periodontitis has been unclear. Here, the effect of Dec2 on the expression of RANKL and on osteoclastogenesis was determined. MATERIAL AND METHODS: Wild-type (WT) and Dec2 knockout (KO) mice as a model for periodontitis were used to assess alveolar bone resorption by microcomputed tomography (CT). Western blot, flow cytometry, quantitative real-time PCR, and immunohistochemical analyses were utilized to detect inflammation and osteoclasts. Luciferase reporter and Chromatin immunoprecipitation (ChIP) assays examined the interaction between Dec2 and RANKL. RESULTS: Micro-CT showed that the alveolar bone resorption of Dec2KO mice was more severe than WT mice after treatment with P. gingivalis. Immunohistochemistry and Tartrate-resistant acid phosphatase staining showed active osteoclast differentiation in Dec2KO mice. There was an increase in CD11b+ F4/80+ and CD4+ RANKL+ T cells in Dec2KO mice treated with P. gingivalis. Moreover, inflammatory and immune markers were expressed at significantly higher levels in gingival mononuclear cells in Dec2KO mice. Furthermore, luciferase reporter and ChIP assays confirmed the direct binding of Dec2 protein to the RANKL gene. CONCLUSION: Dec2 has an immune regulation ability that modulates P. gingivalis-induced periodontitis via RANKL.


Assuntos
Perda do Osso Alveolar , Reabsorção Óssea , Periodontite , Fatores de Transcrição/metabolismo , Perda do Osso Alveolar/diagnóstico por imagem , Animais , Camundongos , Camundongos Knockout , Osteoclastos , Periodontite/diagnóstico por imagem , Periodontite/metabolismo , Ligante RANK/metabolismo , Microtomografia por Raio-X
19.
Comput Biol Med ; 148: 105932, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35964469

RESUMO

High-resolution non-destructive 3D microCT imaging allows the visualization and structural characterization of mineralized cartilage and bone. Deriving statistically relevant quantitative structural information about these tissues, however, requires automated segmentation procedures, mainly because manual contouring is user-biased and time-consuming. Despite the increased spatial resolution in microCT 3D volumes, automatic segmentation of mineralized cartilage versus bone remains non-trivial since they have similar grayscale values. Our work investigates how reliable 2D segmentation masks can be predicted automatically based on a (set of) convolutional neural network(s) trained with a limited number of manually annotated samples. To do that, we compared different strategies to select the 2D samples to annotate and considered ensemble learning and test-time augmentation (TTA) to mitigate the limited accuracy and robustness resulting from the small number of annotated training samples. We show that, for a fixed amount of annotated image samples, 2D microCT slices to annotate should preferably be selected in distinct 3D volumes, at regular intervals, rather than being grouped in adjacent slices of a same 3D volume. Two main lessons are drawn regarding the use of ensembles or TTA instead of a single model. First, ensemble learning is shown to improve segmentation accuracy and to reduce the mean and standard deviation of the absolute errors in cartilage characteristics obtained with different initializations of the neural network training process. In contrast, TTA appears to be unable to improve the model's robustness to unlucky initializations. Second, both TTA and ensembling improved the model's confidence in its predictions and segmentation failure detection.


Assuntos
Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Cartilagem , Aprendizado de Máquina , Imageamento por Ressonância Magnética , Microtomografia por Raio-X
20.
Int J Mol Sci ; 23(16)2022 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-36012308

RESUMO

The ganglioside GD1a has been reported to promote the differentiation of mesenchymal stem cells to osteoblasts in cell culture systems. However, the involvement of gangliosides, including GD1a, in bone formation in vivo remains unknown; therefore, we herein investigated their roles in GM2/GD2 synthase-knockout (GM2/GD2S KO) mice without GD1a. The femoral cancellous bone mass was analyzed using three-dimensional micro-computed tomography. A histomorphometric analysis of bone using hematoxylin and eosin (HE) and tartrate-resistant acid phosphatase was performed to examine bone formation and resorption, respectively. Calcein double labeling was also conducted to evaluate bone formation. Although no significant differences were observed in bone mass or resorption between GM2/GD2S KO mice and wild-type (WT) mice, analyses of the parameters of bone formation using HE staining and calcein double labeling revealed less bone formation in GM2/GD2S KO mice than in WT mice. These results suggest that gangliosides play roles in bone formation.


Assuntos
Gangliosídeos , Osteogênese , Animais , Camundongos , Camundongos Knockout , N-Acetilgalactosaminiltransferases , Osteoblastos , Osteogênese/genética , Microtomografia por Raio-X
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