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1.
J Biomed Nanotechnol ; 16(5): 640-651, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32919484

RESUMO

Cellular starvation induced by glucose oxidase (GOx) had been extensively explored as a potential approach for tumor therapy. However, the therapeutic efficacy suffers daunting challenges due to the unsatisfactory intracellular transportation of GOx molecules. Herein for the first time, hydroxide nanoparticles with unique hollow microstructure (denoted as H-NiAl(OH)x) were designed and synthesized for GOx delivery. In this system, despite its intrinsic degradation properties in acidic tumor microenvironment, Ni2+ ions released during degradation may catalyze a Fenton reaction to induce considerable production of cytotoxic hydroxyl radicals (OH). The cavity of hollow nanocapsules provides large surface area, and favors GOx capsulation and delivery. The findings indicate the intracellular glucose can be effectively consumed by GOx transported, and the reaction products consisting of acid and H2O2 facilitate the OH induction of nanocapsules in a synergistic manner. Both in vitro and in vivo antitumor properties have been consequently achieved by H-NiAl(OH)x/GOx systems. This study offering catalytic nanocapsules based on Ni2+ ions may spark a series of follow-on explorations in constructing drug delivery and therapeutic systems for synergistic tumor treatment.


Assuntos
Nanocápsulas , Nanopartículas , Glucose Oxidase , Peróxido de Hidrogênio , Radical Hidroxila
2.
J Oral Rehabil ; 47(11): 1411-1421, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32841377

RESUMO

OBJECTIVES: Dental rehabilitation post-radiotherapy often requires the consideration of dental implants. However, these are tentatively prescribed due to the concern of hypovascularisation and possible osteoradionecrosis. Hence, the current study assessed the microvasculature of the dento-alveolar bone at implant sites taking into consideration the exact radiotherapy dose received to the region. MATERIALS AND METHODS: Bone cores were taken from nine patients during implant treatment and compared to nine control patients. Specimens were stained using CD31 and digitalised using a high-resolution scanner for qualitative and quantitative assessment of the microvasculature. Monaco® treatment planning system was used to volume the implant site providing mean dose (Dmean ) and maximum dose (Dmax ). RESULTS: A total of 23 bone cores were retrieved for analysis. The cohort had a Dmean of 38.4 Gy (59.6-24.3 Gy). Qualitative analysis identified a clear reduction in the miniscule terminal capillaries and high incidence of obliterated lumens with increasing radiotherapy. Microvasculature density of irradiated patients was markedly reduced (P = .0034) compared to the control group with an inverse correlation to RT doses (P < .0001). Specifically, doses up to 30 Gy appear to preserve sufficient vascularisation (~77% in comparison with control) and tissue architecture. By contrast, exposure to higher doses 40%-61% of the micro-vessels were lost. CONCLUSION: Intensity-modulated radiotherapy doses above 30 Gy identified reduction in microvasculature which is a lower threshold than previously accepted. In pharyngeal cancer patients' doses to the jaw bones often exceed this threshold. Coupled with favourable survival in certain oropharyngeal and nasopharyngeal cancer, dental rehabilitation via implants provides a significant clinical challenge.


Assuntos
Neoplasias Nasofaríngeas , Radioterapia de Intensidade Modulada , Humanos , Microvasos , Pacientes , Dosagem Radioterapêutica
3.
J Tissue Eng ; 11: 2041731419896068, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-35003613

RESUMO

Osteochondral defects remain a major clinical challenge mainly due to the combined damage to the articular cartilage and the underlying bone, and the interface between the two tissues having very different properties. Current treatment modalities have several limitations and drawbacks, with limited capacity of restoration; however, tissue engineering shows promise in improving the clinical outcomes of osteochondral defects. In this study, a novel gradient scaffold has been fabricated, implementing a gradient structure in the design to mimic the anatomical, biological and physicochemical properties of bone and cartilage as closely as possible. Compared with the commonly studied multi-layer scaffolds, the gradient scaffold has the potential to induce a smooth transition between cartilage and bone and avoid any instability at the interface, mimicking the natural structure of the osteochondral tissue. The scaffold comprises a collagen matrix with a gradient distribution of low-crystalline hydroxyapatite particles. Physicochemical analyses confirmed phase and chemical compositions of the gradient scaffold and the distribution of the mineral phase along the gradient scaffold. Mechanical tests confirmed the gradient of stiffness throughout the scaffold, according to its mineral content. The gradient scaffold exhibited good biological performances both in vitro and in vivo. Biological evaluation of the scaffold, in combination with human bone-marrow-derived mesenchymal stem cells, demonstrated that the gradient of composition and stiffness preferentially increased cell proliferation in different sub-regions of the scaffold, according to their high chondrogenic or osteogenic characteristics. The in vivo biocompatibility of the gradient scaffold was confirmed by its subcutaneous implantation in rats. The gradient scaffold was significantly colonised by host cells and minimal foreign body reaction was observed. The scaffold's favourable chemical, physical and biological properties demonstrated that it has good potential as an engineered osteochondral analogue for the regeneration of damaged tissue.

4.
Biomaterials ; 221: 119419, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31421315

RESUMO

An unpredicted side effect of photothermal therapy (PTT) is agitated by hyperthermia which results in damage to healthy tissue. Developing PTT platforms, enabling effective tumor ablation under mild irradiation conditions, is of wide interest, but challenging. Here, we investigated bismuth crystals embedded silica (Bi@SiO2) nanoparticles, loaded with an autophagy inhibitor (chloroquine, CQ). It was found that SiO2 effectively prevented the oxidization of Bi nanocrystals in the physiological environment and could serve as a scatter layer to improve NIR absorption, enabling a high photothermal conversion efficiency (~43%) and excellent photostability. Furthermore, the findings indicated that CQ molecules, delivered intracellularly by the particles, significantly weakened the degradation of autolysosomes by lysosome within the tumor cells, thus inducing suppression effect to autophagy and resistance to photothermia. Both in vitro and in vivo anti-tumor effects were consequently promoted owing to the combined effects enabled by Bi@SiO2-CQ nanoparticles under mild NIR irradiation conditions. This study demonstrates a potential new PTT platform with superior therapeutic efficacy.


Assuntos
Bismuto/química , Nanopartículas/química , Fototerapia/métodos , Dióxido de Silício/química , Animais , Autofagia/fisiologia , Western Blotting , Linhagem Celular Tumoral , Humanos , Camundongos
7.
SLAS Discov ; 24(3): 264-273, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30682324

RESUMO

Endothelial cells (ECs) are widely heterogeneous at the cell level and serve different functions at the vessel and tissue levels. EC-forming colonies derived from induced pluripotent stem cells (iPSC-ECFCs) alongside models such as primary human umbilical vein ECs (HUVECs) are slowly becoming available for research with future applications in cell therapies, disease modeling, and drug discovery. We and others previously described high-content analysis approaches capturing unbiased morphology-based measurements coupled with immunofluorescence and used these for multidimensional reduction and population analysis. Here, we report a tailored workflow to characterize ECs. We acquire images at high resolution with high-magnification water-immersion objectives with Hoechst, vascular endothelial cadherin (VEC), and activated NOTCH staining. We hypothesize that via these key markers alone we would be able to distinguish and assess different EC populations. We used cell population software analysis to phenotype HUVECs and iPSC-ECFCs in the absence or presence of vascular endothelial growth factor (VEGF). To our knowledge, this study presents the first parallel quantitative high-content multiparametric profiling of EC models. Importantly, it highlights a simple strategy to benchmark ECs in different conditions and develop new approaches for biological research and translational applications for regenerative medicine.


Assuntos
Endotélio Vascular/citologia , Biomarcadores/metabolismo , Caderinas/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Receptores Notch/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia
8.
Trends Food Sci Technol ; 78: 155-166, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30100674

RESUMO

Background: Cultured meat forms part of the emerging field of cellular agriculture. Still an early stage field it seeks to deliver products traditionally made through livestock rearing in novel forms that require no, or significantly reduced, animal involvement. Key examples include cultured meat, milk, egg white and leather. Here, we focus upon cultured meat and its technical, socio-political and regulatory challenges and opportunities. Scope and approach: The paper reports the thinking of an interdisciplinary team, all of whom have been active in the field for a number of years. It draws heavily upon the published literature, as well as our own professional experience. This includes ongoing laboratory work to produce cultured meat and over seventy interviews with experts in the area conducted in the social science work. Key findings and conclusions: Cultured meat is a promising, but early stage, technology with key technical challenges including cell source, culture media, mimicking the in-vivo myogenesis environment, animal-derived and synthetic materials, and bioprocessing for commercial-scale production. Analysis of the social context has too readily been reduced to ethics and consumer acceptance, and whilst these are key issues, the importance of the political and institutional forms a cultured meat industry might take must also be recognised, and how ambiguities shape any emergent regulatory system.

9.
Tissue Eng Part A ; 24(23-24): 1775-1783, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29882473

RESUMO

Osteoporosis is characterized by reduced bone mass and aberrant bone microarchitecture, thus increasing susceptibility to fracture due to reduced strength and quality. The aims of this study were to investigate the role of CXCR4 transfected on stem cell homing and osteogenic characteristics in osteopenic rats, particularly elucidating the effect on cell migration. METHODS: Mesenchymal stem cells (MSCs) were harvested from young, and ovariectomized animals and transfected with CXCR4; these cells were administered intravenously in ovariectomized rats. Micro CT and mechanical testing were completed after 12 weeks. RESULTS: Rats injected with young CXCR4 transfected cells had significantly higher bone mineral density (BMD) compared to placebo injected rats (p < 0.05). Rats injected with ovariectomized CXCR4 transfected cells had higher BMD compared to those injected with saline or nontransfected cells (p < 0.04). L4 vertebral stiffness was significantly higher in rats treated with young CXCR4 transfected cells compared to all other groups (p < 0.05). CONCLUSION: CXCR4 genetically modified cells from young and ovariectomized sources improve some aspects of bone formation in the ovariectomized model of osteoporosis and, thus, may play a role in patient treatment regimens.


Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Osteogênese , Osteoporose , Receptores CXCR4 , Animais , Feminino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Osteoporose/genética , Osteoporose/metabolismo , Osteoporose/patologia , Osteoporose/terapia , Ratos , Ratos Wistar , Receptores CXCR4/biossíntese , Receptores CXCR4/genética
10.
Sci Rep ; 7(1): 16293, 2017 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-29176756

RESUMO

Conventional 3D bioprinting allows fabrication of 3D scaffolds for biomedical applications. In this contribution we present a cryogenic 3D printing method able to produce stable 3D structures by utilising the liquid to solid phase change of a composite hydrogel (CH) ink. This is achieved by rapidly cooling the ink solution below its freezing point using solid carbon dioxide (CO2) in an isopropanol bath. The setup was able to successfully create 3D complex geometrical structures, with an average compressive stiffness of O(1) kPa (0.49 ± 0.04 kPa stress at 30% compressive strain) and therefore mimics the mechanical properties of the softest tissues found in the human body (e.g. brain and lung). The method was further validated by showing that the 3D printed material was well matched to the cast-moulded equivalent in terms of mechanical properties and microstructure. A preliminary biological evaluation on the 3D printed material, coated with collagen type I, poly-L-lysine and gelatine, was performed by seeding human dermal fibroblasts. Cells showed good attachment and viability on the collagen-coated 3D printed CH. This greatly widens the range of applications for the cryogenically 3D printed CH structures, from soft tissue phantoms for surgical training and simulations to mechanobiology and tissue engineering.

11.
J Tissue Eng ; 5: 2041731414536572, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24904727

RESUMO

Tissue engineering-based bone grafts are emerging as a viable alternative treatment modality to repair and regenerate tissues damaged as a result of disease or injury. The choice of the biomaterial component is a critical determinant of the success of the graft or scaffold; essentially, it must induce and allow native tissue integration, and most importantly mimic the hierarchical structure of the native bone. Calcium phosphate bioceramics are widely used in orthopaedics and dentistry applications due to their similarity to bone mineral and their ability to induce a favourable biological response. One such material is monetite, which is biocompatible, osteoconductive and has the ability to be resorbed under physiological conditions. The osteoinductive properties of monetite in vivo are known; however, little is known of the direct effect on osteoinduction of human mesenchymal stem cells in vitro. In this study, we evaluated the potential of monetite to induce and sustain human mesenchymal stem cells towards osteogenic differentiation. Human mesenchymal stem cells were seeded on the monetite scaffold in the absence of differentiating factors for up to 28 days. The gene expression profile of bone-specific markers in cells on monetite scaffold was compared to the control material hydroxyapatite. At day 14, we observed a marked increase in alkaline phosphatase, osteocalcin and osteonectin expressions. This study provides evidence of a suitable material that has potential properties to be used as a tissue engineering scaffold.

12.
J Craniomaxillofac Surg ; 42(6): 863-9, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24485270

RESUMO

The investigation aims to assess the reconstruction of critical-size mandibular bone defects in rabbits using beta-Tricalcium Phosphate (ß-TCP) scaffolding loaded with stem cells. A 20 mm-long mandibular osteoperiosteal continuity defect was created in 8 New Zealand rabbits and filled with ß-TCP scaffolding. In 6 cases bone marrow stem cells (BMSCs) harvested, and enriched, from the posterior iliac crest of the same rabbit were seeded into the scaffolding, while a scaffold was used alone in two cases chosen at random. Radiographic analysis was carried out immediately following surgery and 4, 8 and 12 weeks postoperatively. Cone Beam CT (CBCT) scanning, biomechanical testing and histology assessments were carried out on the explanted mandibles three months postoperatively. The radiography showed minimal new bone formation in all the cases, with significant amounts of undegraded scaffold material visible. Sporadic areas of bone formation were seen, these did not bridge the gap of the created surgical defect. The mechanical properties of the regenerated bone were of an inferior quality when compared with that of the contralateral non-operated side. The addition of BMSCs to the biodegradable ß-TCP scaffold did not improve reconstruction of the created mandibular defect. Despite successful aspiration and culture of BMSCs, the survival of these cells in vivo was questionable.


Assuntos
Materiais Biocompatíveis/química , Regeneração Óssea/fisiologia , Fosfatos de Cálcio/química , Doenças Mandibulares/cirurgia , Células-Tronco Mesenquimais/fisiologia , Tecidos Suporte/química , Animais , Fenômenos Biomecânicos , Técnicas de Cultura de Células , Forma Celular , Sobrevivência Celular/fisiologia , Tomografia Computadorizada de Feixe Cônico/métodos , Meios de Cultura , Masculino , Mandíbula/patologia , Doenças Mandibulares/patologia , Osteogênese/fisiologia , Coelhos , Distribuição Aleatória , Estresse Mecânico , Fatores de Tempo , Engenharia Tecidual/métodos
13.
J R Soc Interface ; 11(93): 20140004, 2014 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-24478288

RESUMO

Bone cells (osteoblasts) produce a collagen-rich matrix called osteoid, which is mineralized extracellularly by nanosized calcium phosphate (CaP). Synthetically produced CaP nanoparticles (NPs) have great potential for clinical application. However few studies have compared the effect of CaP NPs with different properties, such as shape and aspect ratio, on the survival and behaviour of active bone-producing cells, such as primary human osteoblasts (HOBs). This study aimed to investigate the biocompatibility and ultrastructural effects of two differently shaped hydroxyapatite [Ca10(PO4)6(OH)2] nanoparticles (HA NPs), round- (aspect ratio 2.12, AR2) and rice-shaped (aspect ratio 3.79, AR4). The ultrastructural response and initial extracellular matrix (ECM) formation of HOBs to HA NPs were observed, as well as matrix vesicle release. A transmission electron microscopy (TEM)-based X-ray microanalytical technique was used to measure cytoplasmic ion levels, including calcium (Ca), phosphorus (P), sodium (Na) and potassium (K). K/Na ratios were used as a measure of cell viability. Following HA NP stimulation, all measured cytoplasmic ion levels increased. AR2 NPs had a greater osteogenic effect on osteoblasts compared with AR4 NPs, including alkaline phosphatase activity and matrix vesicle release. However, they produced only a moderate increase in intracellular Ca and P levels compared with AR4. This suggests that particular Ca and P concentrations may be required for, or indicative of, optimal osteoblast activity. Cell viability, as measured by Na and K microanalysis, was best maintained in AR2. Initial formation of osteoblast ECM was altered in the presence of either HA NP, and immuno-TEM identified fibronectin and matrilin-3 as two ECM proteins affected. Matrilin-3 is here described for the first time as being expressed by cultured osteoblasts. In summary, this novel and in-depth study has demonstrated that HA NP shape can influence a range of different parameters related to osteoblast viability and activity.


Assuntos
Durapatita/farmacologia , Teste de Materiais , Nanopartículas/química , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citoplasma/metabolismo , Durapatita/química , Fibronectinas/metabolismo , Humanos , Íons/metabolismo , Proteínas Matrilinas/metabolismo , Microscopia Eletrônica de Transmissão , Nanopartículas/ultraestrutura , Osteoblastos/ultraestrutura
14.
Mater Sci Eng C Mater Biol Appl ; 36: 206-14, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24433905

RESUMO

A multi-step sol-gel process was employed to synthesize bioactive glass (BG) nanoparticles. Transmission electron microscopy (TEM) revealed that the BG nanoparticles were spherical and ranged from 30 to 60 nm in diameter. In vitro reactivity of the BG nanoparticles was tested in phosphate buffer saline (PBS), Tris-buffer (TRIS), simulated body fluid (SBF), and Dulbecco's modified Eagle's medium (DMEM), in comparison with similar sized hydroxyapatite (HA) and silicon substituted HA (SiHA) nanoparticles. Bioactivity of the BG nanoparticles was confirmed through Fourier transform infrared spectroscopy (FTIR) analysis. It was found that bone-like apatite was formed after immersion in SBF at 7 days. Solutions containing BG nanoparticles were slightly more alkaline than HA and SiHA, suggesting that a more rapid apatite formation on BG was related to solution-mediated dissolution. Primary human osteoblast (HOB) cell model was used to evaluate biological responses to BG nanoparticles. Lactate dehydrogenase (LDH) cytotoxicity assay showed that HOB cells were not adversely affected by the BG nanoparticles throughout the 7day test period. Interestingly, MTS assay results showed an enhancement in cell proliferation in the presence of BG when compared to HA and SiHA nanoparticles. Particularly, statistically significant (p<0.05) alkaline phosphatase (ALP) activity of HOB cells was found on the culture containing BG nanoparticles, suggesting that the cell differentiation might be promoted by BG. Real-time quantitative PCR analysis (qPCR) further confirmed this finding, as a significantly higher level of RUNX2 gene expression was recorded on the cells cultured in the presence of BG nanoparticles when compared to those with HA and SiHA.


Assuntos
Osso e Ossos/fisiologia , Vidro/química , Nanopartículas/química , Osteoblastos/citologia , Transição de Fase , Engenharia Tecidual/métodos , Fosfatase Alcalina/metabolismo , Morte Celular , Proliferação de Células , Forma Celular , Durapatita/química , Humanos , Concentração de Íons de Hidrogênio , Nanopartículas/ultraestrutura , Osteoblastos/enzimologia , Osteoblastos/ultraestrutura , Osteogênese , Tamanho da Partícula , Reação em Cadeia da Polimerase em Tempo Real , Silício/química , Soluções , Espectrometria por Raios X , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios X
15.
Br J Oral Maxillofac Surg ; 52(1): 7-15, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23601833

RESUMO

Tissue engineering is a rapidly advancing discipline that combines the attributes of biochemical and biomaterial engineering with cell transplantation to create bio-artificial tissues and organs. For the oral and maxillofacial surgeon, the reconstruction of maxillofacial defects in hard and soft tissues is an ongoing challenge. While autologous grafts and vascularised free flaps are the current gold standard, they are not without complications at both the donor and reconstructed sites. Tissue engineering, which aims to create tissue-matched, prefabricated, prevascularised bony or soft tissue composite grafts, or both, therefore has the potential to revolutionise practice in maxillofacial surgery. We review the technology of tissue engineering and its current and future applications within the specialty, and discuss contemporary obstacles yet to be overcome.


Assuntos
Procedimentos Cirúrgicos Bucais/métodos , Engenharia Tecidual/métodos , Autoenxertos/transplante , Tecnologia Biomédica , Materiais Biomiméticos/uso terapêutico , Transplante de Células , Retalhos de Tecido Biológico/transplante , Humanos , Tecidos Suporte , Sítio Doador de Transplante/cirurgia
16.
J Periodontol ; 85(2): 298-307, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23786402

RESUMO

BACKGROUND: Calcium phosphate-based materials have been widely used as bone substitutes and more recently are being exploited together with growth factors as bone tissue engineering scaffolds regulating cell behavior. The aim of this study is to evaluate the in vitro and in vivo response to a newly developed calcium metaphosphate (CMP) bone graft, with and without bone-stimulating growth factor. METHODS: Porous scaffolds of CMP were developed and extensively tested in vitro. Subsequently, CMP grafts with osteogenic protein-1 (OP-1) (test) and without OP-1 (control) were implanted into experimental rabbit maxillary bone defects. Animals were sacrificed at 2, 4, and 8 weeks, and samples were examined with microcomputed tomography (micro-CT) and processed for histomorphometric analysis. RESULTS: At 8 weeks, the scaffolds containing OP-1 induced greater bone formation (P = 0.018) than CMP alone, based on histomorphometric evaluation (percentage bone area: test: 57.1 ± 5.6; control: 49.4 ± 7.7) and micro-CT analysis (percentage bone volume density: test: 63.46 ± 5.61; control: 51.20 ± 6.71). Thus, these data indicated that both test and control CMP grafts showed a good degree of bone formation. Furthermore, the CMP materials showed signs of resorption from 4 weeks, and no graft materials were observed at 8 weeks. CONCLUSIONS: In vitro, the OP-1 loaded graft demonstrated a release profile and bioactivity over a 28-day period. In vivo testing confirmed enhanced bone formation of the OP-1 loaded graft after 8 weeks of healing.


Assuntos
Proteína Morfogenética Óssea 7/uso terapêutico , Fosfatos de Cálcio/química , Doenças Maxilares/cirurgia , Tecidos Suporte/química , Implantes Absorvíveis , Fosfatase Alcalina/análise , Fosfatase Alcalina/efeitos dos fármacos , Animais , Densidade Óssea/efeitos dos fármacos , Proteína Morfogenética Óssea 7/química , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Corantes , Difusão , Feminino , Humanos , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Doenças Maxilares/patologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteocalcina/análise , Osteocalcina/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Coelhos , Sais de Tetrazólio , Tiazóis , Fatores de Tempo , Microtomografia por Raio-X/métodos
17.
Tissue Eng Part A ; 19(21-22): 2426-38, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23968499

RESUMO

INTRODUCTION: The aim of the study reported here was to investigate the molecular responses of human mesenchymal stem cells (MSC) to loading with a model that attempts to closely mimic the physiological mechanical loading of bone, using monetite calcium phosphate (CaP) scaffolds to mimic the biomechanical properties of bone and a bioreactor to induce appropriate load and strain. METHODS: Human MSCs were seeded onto CaP scaffolds and subjected to a pulsating compressive force of 5.5±4.5 N at a frequency of 0.1 Hz. Early molecular responses to mechanical loading were assessed by microarray and quantitative reverse transcription-polymerase chain reaction and activation of signal transduction cascades was evaluated by western blotting analysis. RESULTS: The maximum mechanical strain on cell/scaffolds was calculated at around 0.4%. After 2 h of loading, a total of 100 genes were differentially expressed. The largest cluster of genes activated with 2 h stimulation was the regulator of transcription, and it included FOSB. There were also changes in genes involved in cell cycle and regulation of protein kinase cascades. When cells were rested for 6 h after mechanical stimulation, gene expression returned to normal. Further resting for a total of 22 h induced upregulation of 63 totally distinct genes that were mainly involved in cell surface receptor signal transduction and regulation of metabolic and cell division processes. In addition, the osteogenic transcription factor RUNX-2 was upregulated. Twenty-four hours of persistent loading also markedly induced osterix expression. Mechanical loading resulted in upregulation of Erk1/2 phosphorylation and the gene expression study identified a number of possible genes (SPRY2, RIPK1, SPRED2, SERTAD1, TRIB1, and RAPGEF2) that may regulate this process. CONCLUSION: The results suggest that mechanical loading activates a small number of immediate-early response genes that are mainly associated with transcriptional regulation, which subsequently results in activation of a wider group of genes including those associated with osteoblast proliferation and differentiation. The results provide a valuable insight into molecular events and signal transduction pathways involved in the regulation of MSC osteogenic differentiation in response to a physiological level of mechanical stimulation.


Assuntos
Fosfatos de Cálcio/química , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Tecidos Suporte/química , Técnicas de Cultura de Células , Sobrevivência Celular/fisiologia , Células Cultivadas , Humanos , Estresse Mecânico
18.
Macromol Biosci ; 13(7): 851-9, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23765615

RESUMO

An immortalized human dental pulp stem cell (DPSC) line of an odontoblastic phenotype is established to circumvent the normal programmed senescence and to maintain the cell line's usefulness as a tool for further study of cellular activity. DPSCs are isolated from human dental pulp tissues and transfected using hTERT. The influence of this process on the DPSC phenotype and the mRNA expression of oncogenes involved in cellular senescence is investigated. The results reveal an absence of altered DPSC morphology and phenotype following the exogenous introduction of the hTERT gene, which is coupled with a significant reduction in p16 mRNA expression. This provides insight into how to circumvent in vitro dental pulp stem cell death following the exogenous introduction of hTERT.


Assuntos
Polpa Dentária/citologia , Odontoblastos/metabolismo , Células-Tronco/citologia , Telomerase/genética , Linhagem Celular , Proliferação de Células , Senescência Celular , Expressão Gênica , Genes p16 , Humanos , Odontoblastos/citologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transdução Genética
19.
J Craniomaxillofac Surg ; 40(8): e461-9, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22507295

RESUMO

This investigation assesses the histological, radiographic and mechanical properties of regenerated bone in a unilateral critical-size osteoperiosteal mandibular continuity defect in the rabbit model, following the application of beta-tricalcium phosphate (ß-TCP) scaffolding and recombinant human bone morphogenetic protein 7 (rhBMP-7). The study was carried out on nine cases; in six cases the critical-size defect was filled with rhBMP-7 in the ß-TCP scaffolding, and in three cases the ß-TCP was used alone. The cases were sacrificed 3 months post-operatively. Histologically the overall mean of the percentage of regenerated bone volume in the cases that received rhBMP-7 was 29.41% ± 6.25%, which was considerably greater than the 6.35% ± 3.08% in the cases treated with ß-TCP alone. Mechanical testing of the cases treated with rhBMP-7 gave failure moments (55 mNm-2.040 Nm) that were consistently greater than those treated with ß-TCP alone (0 mNm-48 mNm). In some cases the mechanical properties of the regenerated bone were comparable to those of untreated bone. RhBMP-7 in prefabricated ß-TCP scaffolding appeared, radiographically and histologically, to be an effective method for bone regeneration in mandibular critical-size defects in the rabbit model. This points towards possible future clinical applications.


Assuntos
Materiais Biocompatíveis , Proteína Morfogenética Óssea 7/uso terapêutico , Fosfatos de Cálcio , Reconstrução Mandibular/métodos , Procedimentos Cirúrgicos Reconstrutivos/métodos , Tecidos Suporte , Animais , Materiais Biocompatíveis/química , Fenômenos Biomecânicos , Densidade Óssea/efeitos dos fármacos , Densidade Óssea/fisiologia , Regeneração Óssea/efeitos dos fármacos , Regeneração Óssea/fisiologia , Remodelação Óssea/efeitos dos fármacos , Remodelação Óssea/fisiologia , Fosfatos de Cálcio/química , Tomografia Computadorizada de Feixe Cônico/métodos , Modelos Animais de Doenças , Humanos , Mandíbula/patologia , Mandíbula/cirurgia , Doenças Mandibulares/patologia , Doenças Mandibulares/cirurgia , Fraturas Mandibulares/fisiopatologia , Reconstrução Mandibular/instrumentação , Coelhos , Procedimentos Cirúrgicos Reconstrutivos/instrumentação , Estresse Mecânico , Fatores de Tempo , Tecidos Suporte/química
20.
Cell Cycle ; 10(22): 3912-9, 2011 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-22067611

RESUMO

INTRODUCTION: Residing within human dental pulp are cells of an ectomesenchymal origin which have the potential to differentiate into odontoblast-like cells. These cells have a limited growth potential owing to the effects of cell senescence. This study examines the effects of immortalizing odontoblast-like cells on cell proliferation and mineralization by comparing transformed dental pulp stem cells (tDPSCs) and non-transformed dental pulp stem cells (nDPSCs). RESULTS: With the exogenous expression of hTERT, tDPSCs maintained a continued expression of odontogenic markers for cell proliferation and mineralization (ALP, COL-1, DMP-1, DSPP, OCN amd OPN)as did nDPScs. Oncoprotein expression was seen in both groups except for a noted absence of p16 in the tDPSCs. nDPSCs also showed lower levels of total ALP and DNA activity in comparison to tDPSCs when assayed as well as low telomerase activity readings. METHODS: Using a retroviral vector, exogenous human telomerase reverse transcriptase (hTERT) was expressed in tDPSCs. Both cell groups were cultured and their telomerase activities is determined using a telomerase quantification assay. Also examined were the expression of genes involved in proliferation and mineralization such as human alkaline phosphatase (ALP), ß-actin, collagen 1 (col-1), core binding factor (cbfa-1), dentin matrix protein (DMP-1), dentin sialophosphoprotein (DSPP), GAPDH, hTERT, osteocalcin (OCN), osteopontin (OPN) as well as oncoproteins involved in senescence (p16, p21 and p53) using RT-PCR. DNA and alkaline phosphatase activity was assayed in both cell groups. CONCLUSIONS: These results indicate maintainance of odontoblast-like differentiation characteristics after retroviral transformation with hTERT and suggest a possible link with a reduced p16 expression.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Polpa Dentária/metabolismo , Telomerase/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Biomarcadores/análise , Biomarcadores/metabolismo , Diferenciação Celular , Proliferação de Células , Colorimetria , Inibidor p16 de Quinase Dependente de Ciclina/análise , Citoesqueleto/ultraestrutura , Polpa Dentária/citologia , Polpa Dentária/ultraestrutura , Humanos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telomerase/farmacologia , Transdução Genética , Proteína Supressora de Tumor p53/análise , beta-Galactosidase/análise
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