Gene-modified BMSCs encapsulated with carboxymethyl cellulose facilitate osteogenesis in vitro and in vivo

Critical size bone defects are one of the most serious complications in orthopedics due to the lack of effective osteogenesis treatment. We fabricated carboxymethyl cellulose with phenol moieties (CMC-ph) microcapsules loaded with gene-modified rat bone mesenchymal stem cells (rBMSCs) that secrete hBMP2 following doxycycline (DOX) induction. The results showed that the morphology of microcapsules was spherical, and their diameters have equally distributed in the range of 100–150 μm; the viability of rBMSCs was unchanged over time. Through real-time PCR and Western blot analyses, the rBMSCs in microcapsules were found to secrete hBMP2 and to have upregulated mRNA and protein expression of osteogenesis-related genes in vitro and in vivo. Furthermore, the in vivo results suggested that the group with the middle concentration of cells expressed the highest amount of osteogenic protein over time. In this study, we showed that gene-modified rBMSCs in CMC-ph microcapsules had good morphology and viability. The BMP2–BMSCs/CMC-Ph microcapsule system could upregulate osteogenic mRNA and protein in vitro and in vivo. Further analysis demonstrated that the medium concentration of cells had a suitable density for transplantation in nude mice. Therefore, BMP2–BMSCs/CMC-Ph microcapsule constructs have potential for bone regeneration in vivo.

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A preliminary study on the evaluation of a novel gene delivery vector-TACS in vitro and in vivo via co-expressing hVEGF and hBMP genes to repair critical-size rabbit bone defects

African Journal of Pharmacy and Pharmacology ◽

10.5897/ajpp11.822 ◽

2012 ◽

Vol 6 (5) ◽

Author(s):

Yu Wenyuan

Keyword(s):

Gene Delivery ◽

Critical Size ◽

Bone Defects ◽

Gene Delivery Vector ◽

Novel Gene ◽

Delivery Vector ◽

Rabbit Bone ◽

Preliminary Study

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Treatment of Critical-Size Femoral Bone Defects with Chitosan Scaffolds Produced by a Novel Process from Textile Engineering

Biomedicines ◽

10.3390/biomedicines9081015 ◽

2021 ◽

Vol 9 (8) ◽

pp. 1015

Author(s):

Bruno Zwingenberger ◽

Corina Vater ◽

Roland L. Bell ◽

Julia Bolte ◽

Elisabeth Mehnert ◽

...

Keyword(s):

Mechanical Stability ◽

Critical Size ◽

Bone Allograft ◽

Bone Defects ◽

Lower Grade ◽

Femoral Bone ◽

Human Cancellous Bone ◽

Human Telomerase

The purpose of this study was to investigate, in vitro and in vivo, the suitability of chitosan (CHS) scaffolds produced by the net-shape-nonwoven (NSN) technology, for use as bone graft substitutes in a critical-size femoral bone defect in rats. For in vitro investigations, scaffolds made of CHS, mineralized collagen (MCM), or human cancellous bone allograft (CBA) were seeded with human telomerase-immortalized mesenchymal stromal cells (hTERT-MSC), incubated for 14 days, and thereafter evaluated for proliferation and osteogenic differentiation. In vivo, CHS, MCM and CBA scaffolds were implanted into 5 mm critical-size femoral bone defects in rats. After 12 weeks, the volume of newly formed bone was determined by microcomputed tomography (µCT), while the degree of defect healing, as well as vascularization and the number of osteoblasts and osteoclasts, was evaluated histologically. In vitro, CHS scaffolds showed significantly higher osteogenic properties, whereas treatment with CHS, in vivo, led to a lower grade of bone-healing compared to CBA and MCM. While chitosan offers a completely new field of scaffold production by fibers, these scaffolds will have to be improved in the future, regarding mechanical stability and osteoconductivity.

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The Development of Homologous (Canine/Anti-Canine) Antibodies in Dogs with Haemophilia A (Factor VIII Deficiency): A Ten-Year Longitudinal Study

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651541 ◽

1993 ◽

Vol 69 (01) ◽

pp. 021-024 ◽

Cited By ~ 20

Author(s):

Shawn Tinlin ◽

Sandra Webster ◽

Alan R Giles

Keyword(s):

Factor Viii ◽

Canine Model ◽

Significant Inhibition ◽

Human Condition ◽

Haemophilia A ◽

Variable Expression ◽

The Family ◽

Over Time

SummaryThe development of inhibitors to factor VIII in patients with haemophilia A remains as a serious complication of replacement therapy. An apparently analogous condition has been described in a canine model of haemophilia A (Giles et al., Blood 1984; 63:451). These animals and their relatives have now been followed for 10 years. The observation that the propensity for inhibitor development was not related to the ancestral factor VIII gene has been confirmed by the demonstration of vertical transmission through three generations of the segment of the family related to a normal (non-carrier) female that was introduced for breeding purposes. Haemophilic animals unrelated to this animal have not developed functionally significant factor VIII inhibitors despite intensive factor VIII replacement. Two animals have shown occasional laboratory evidence of factor VIII inhibition but this has not been translated into clinical significant inhibition in vivo as assessed by clinical response and F.VIII recovery and survival characteristics. Substantial heterogeneity of inhibitor expression both in vitro and in vivo has been observed between animals and in individual animals over time. Spontaneous loss of inhibitors has been observed without any therapies designed to induce tolerance, etc., being instituted. There is also phenotypic evidence of polyclonality of the immune response with variable expression over time in a given animal. These observations may have relevance to the human condition both in determining the pathogenetic factors involved in this condition and in highlighting the heterogeneity of its expression which suggests the need for caution in the interpretation of the outcome of interventions designed to modulate inhibitor activity.

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Faculty Opinions recommendation of In vivo investigation of tissue-engineered periosteum for the repair of allogeneic critical size bone defects in rabbits.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.727723479.793545144 ◽

2018 ◽

Author(s):

Warren Grayson

Keyword(s):

Critical Size ◽

Bone Defects

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Upregulation of ETV2 Expression Promotes Endothelial Differentiation of Human Dental Pulp Stem Cells

Cell Transplantation ◽

10.1177/0963689720978739 ◽

2021 ◽

Vol 30 ◽

pp. 096368972097873

Author(s):

Jing Li ◽

Youming Zhu ◽

Na Li ◽

Tao Wu ◽

Xianyu Zheng ◽

...

Keyword(s):

Protein Expression ◽

Dental Pulp ◽

Tube Formation ◽

Endothelial Differentiation ◽

Cell Source ◽

Lentiviral Infection ◽

Mrna And Protein Expression ◽

Human Dental Pulp

The lack of vasculogenesis often hampers the survivability and integration of newly engineered tissue grafts within the host. Autologous endothelial cells (ECs) are an ideal cell source for neovascularization, but they are limited by their scarcity, lack of proliferative capacity, and donor site morbidity upon isolation. The objective of this study was to determine whether differentiation of human dental pulp stem cells (DPSCs) into the endothelial lineage can be enhanced by recombinant ETV2 overexpression. DPSCs were extracted from fresh dental pulp tissues. ETV2 overexpression in DPSCs was achieved by lentiviral infection and cellular morphological changes were evaluated. The mRNA and protein expression levels of endothelial-specific markers were assessed through quantitative real-time polymerase chain reaction, western blot, immunofluorescence staining, and flow cytometry. The tube formation assay and Matrigel plug assay were also performed to evaluate the angiogenic potential of the ETV2-transduced cells in vitro and in vivo, respectively. Additionally, proteomic analysis was performed to analyze global changes in protein expression following ETV2 overexpression. After lentiviral infection, ETV2-overexpressing DPSCs showed endothelial-like morphology. Compared with control DPSCs, significantly higher mRNA and protein expression levels of endothelial-specific genes, including CD31, VE-Cadherin, VEGFR1, and VEGFR2, were detected in ETV2-overexpressing DPSCs. Moreover, ETV2 overexpression enhanced capillary-like tube formation on Matrigel in vitro, as well as neovascularization in vivo. In addition, comparative proteomic profiling showed that ETV2 overexpression upregulated the expression of vascular endothelial growth factor (VEGF) receptors, which was indicative of increased VEGF signaling. Taken together, our results indicate that ETV2 overexpression significantly enhanced the endothelial differentiation of DPSCs. Thus, this study shows that DPSCs can be a promising candidate cell source for tissue engineering applications.

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Abstract 5402: Two Photon Microscopy Measurements Of Sub-epicardial Sarcomere Length In Perfused Rat Hearts

Circulation ◽

10.1161/circ.118.suppl_18.s_543-c ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Patrizia Camelliti ◽

Gil Bub ◽

Daniel J Stuckey ◽

Christian Bollensdorff ◽

Damian J Tyler ◽

...

Keyword(s):

Sarcomere Length ◽

Left Ventricular ◽

Model Systems ◽

Future Research ◽

Fundamental Parameter ◽

Rat Hearts ◽

Perfused Hearts ◽

Over Time

Sarcomere length (SL) is a fundamental parameter underlying the Frank Starling relation in the heart, as it offers an absolute representation of myocardial stretch. Previous studies addressed the Frank Starling relation by measuring SL in isolated myocytes or muscle strips. Here, we report first data obtained using a novel technique to measure sub-epicardial SL in perfused hearts. Rat hearts were Langendorff perfused (normal Tyrode solution) at a constant pressure of 90mmHg, labeled with the fluorescent membrane marker di-4-ANEPPS, and then arrested with high-K + Tyrode for either 2-photon microscopy (n=4) or MRI (n=4). Image analysis software was developed to extract SL at the cell level from >1,400 2-photon images (Fig 1 ) and correct for cell angle. SL increased by 10±2 % between 30 and 80 min of perfusion (1.98±0.04 to 2.17±0.03 μm; p<0.05; Fig 1 ). Measurements of left ventricular myocardial volume (LVMV) were made in vivo and in perfused hearts using 3D MRI. LVMV increased by 24±7% from in vivo to 30 min of perfusion, and by 11±3 % between 30 and 90 min (539±35; 664±44; 737±49 mm 3 , respectively; p<0.05; Fig 1 ). We show that SL can be measured in isolated perfused hearts. The method allowed monitoring of changes in SL over time, and showed that SL and LVMV increase to a similar extent during 30–80 min perfusion with crystalloid solution, probably due to tissue oedema. This result, together with the increase in LVMV during the first 30 min, highlights the pronounced differences between in vivo , in situ , and in vitro model systems for studies of cardiac physiology and mechanics. Future research will compare changes in SL in healthy hearts and disease models involving contractile dysfunction. Figure 1: Left: 2-photon microscopy image of di-4-ANEPPS labeled myocardium. Right: SL and LVMV changes over time.

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Biocompatibility and Bone Formation of Flexible, Cotton Wool-like PLGA/Calcium Phosphate Nanocomposites in Sheep

The Open Orthopaedics Journal ◽

10.2174/1874325001105010063 ◽

2011 ◽

Vol 5 (1) ◽

pp. 63-71 ◽

Cited By ~ 35

Author(s):

Oliver D Schneider ◽

Dirk Mohn ◽

Roland Fuhrer ◽

Karina Klein ◽

Käthi Kämpf ◽

...

Keyword(s):

Calcium Phosphate ◽

Bone Formation ◽

Antimicrobial Properties ◽

Drill Hole ◽

Bone Defects ◽

Long Bone ◽

Minimal Amount ◽

Cotton Wool

Background: The purpose of this preliminary study was to assess the in vivo performance of synthetic, cotton wool-like nanocomposites consisting of a biodegradable poly(lactide-co-glycolide) fibrous matrix and containing either calcium phosphate nanoparticles (PLGA/CaP 60:40) or silver doped CaP nanoparticles (PLGA/Ag-CaP 60:40). Besides its extraordinary in vitro bioactivity the latter biomaterial (0.4 wt% total silver concentration) provides additional antimicrobial properties for treating bone defects exposed to microorganisms. Materials and Methods: Both flexible artificial bone substitutes were implanted into totally 16 epiphyseal and metaphyseal drill hole defects of long bone in sheep and followed for 8 weeks. Histological and histomorphological analyses were conducted to evaluate the biocompatibility and bone formation applying a score system. The influence of silver on the in vivo performance was further investigated. Results: Semi-quantitative evaluation of histology sections showed for both implant materials an excellent biocompatibility and bone healing with no resorption in the adjacent bone. No signs of inflammation were detectable, either macroscopically or microscopically, as was evident in 5 µm plastic sections by the minimal amount of inflammatory cells. The fibrous biomaterials enabled bone formation directly in the centre of the former defect. The area fraction of new bone formation as determined histomorphometrically after 8 weeks implantation was very similar with 20.5 ± 11.2 % and 22.5 ± 9.2 % for PLGA/CaP and PLGA/Ag-CaP, respectively. Conclusions: The cotton wool-like bone substitute material is easily applicable, biocompatible and might be beneficial in minimal invasive surgery for treating bone defects.

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Mn Inhibits GSH Synthesis via Downregulation of Neuronal EAAC1 and Astrocytic xCT to Cause Oxidative Damage in the Striatum of Mice

Oxidative Medicine and Cellular Longevity ◽

10.1155/2018/4235695 ◽

2018 ◽

Vol 2018 ◽

pp. 1-15 ◽

Cited By ~ 7

Author(s):

Xinxin Yang ◽

Haibo Yang ◽

Fengdi Wu ◽

Zhipeng Qi ◽

Jiashuo Li ◽

...

Keyword(s):

Oxidative Stress ◽

Oxidative Damage ◽

Dependent Manner ◽

Protein Levels ◽

Key Factor ◽

Protein Sulfhydryl ◽

Mrna And Protein Expression ◽

The Brain

Excessive manganese (Mn) can accumulate in the striatum of the brain following overexposure. Oxidative stress is a well-recognized mechanism in Mn-induced neurotoxicity. It has been proven that glutathione (GSH) depletion is a key factor in oxidative damage during Mn exposure. However, no study has focused on the dysfunction of GSH synthesis-induced oxidative stress in the brain during Mn exposure. The objective of the present study was to explore the mechanism of Mn disruption of GSH synthesis via EAAC1 and xCT in vitro and in vivo. Primary neurons and astrocytes were cultured and treated with different doses of Mn to observe the state of cells and levels of GSH and reactive oxygen species (ROS) and measure mRNA and protein expression of EAAC1 and xCT. Mice were randomly divided into seven groups, which received saline, 12.5, 25, and 50 mg/kg MnCl2, 500 mg/kg AAH (EAAC1 inhibitor) + 50 mg/kg MnCl2, 75 mg/kg SSZ (xCT inhibitor) + 50 mg/kg MnCl2, and 100 mg/kg NAC (GSH rescuer) + 50 mg/kg MnCl2 once daily for two weeks. Then, levels of EAAC1, xCT, ROS, GSH, malondialdehyde (MDA), protein sulfhydryl, carbonyl, 8-hydroxy-2-deoxyguanosine (8-OHdG), and morphological and ultrastructural features in the striatum of mice were measured. Mn reduced protein levels, mRNA expression, and immunofluorescence intensity of EAAC1 and xCT. Mn also decreased the level of GSH, sulfhydryl, and increased ROS, MDA, 8-OHdG, and carbonyl in a dose-dependent manner. Injury-related pathological and ultrastructure changes in the striatum of mice were significantly present. In conclusion, excessive exposure to Mn disrupts GSH synthesis through inhibition of EAAC1 and xCT to trigger oxidative damage in the striatum.

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