RESUMO
BACKGROUND: Oral lichen planus (OLP) is a chronic inflammatory disorder with increased risk for malignant transformation. Biomarker validation is a pivotal step in moving newly discovered biomarkers towards clinical implementation. We performed a systematic review of studies on biomarkers related to OLP, wherein biomarkers have been described in at least two independent studies. Our aim was to determine whether any of these biomarkers might be promising in predicting the increased risk of malignant transformation of OLP. MATERIAL AND METHODS: We searched the following databases until August 2021: PUBMED, EMBASE, and Web of Science. Due to high heterogeneity, a qualitative rather than quantitative assessment was conducted. Only proteins that consistently showed a significantly high level of expression in neoplastic tissues versus OLP in two or more publications were considered as promising markers. RESULTS: Initial database researches identified 1671, of which 24 articles were included in the final analysis. The most frequently reported proteins were p53, Bcl-2 and Ki-67, though there were controversies. PCNA and P21 were the only proteins that showed consistent evidence of clinical usefulness as cancer predictors to be considered as promising markers. Extensive methodological variations in the evaluation of expressions and statistical analyses of the included markers were observed, which hampered comparisons of the results. CONCLUSIONS: Multiple levels of heterogeneity with a scarcity of high-quality studies were identified. PCNA and P21 were identified as promising predictive markers for evaluating the risk of malignant transformation of OLP, but they require further validation. The focus of future research on validation of predictive biomarkers of OLP should be considered as a high priority because it will accelerate the introduction of newly discovered markers into the clinical setting.
Assuntos
Líquen Plano Bucal , Neoplasias Bucais , Biomarcadores , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Humanos , Imuno-Histoquímica , Líquen Plano Bucal/patologia , Neoplasias Bucais/patologia , Antígeno Nuclear de Célula em ProliferaçãoRESUMO
BACKGROUND: Alveolar cleft grafting is a necessary procedure to restore bone defects. Randomized clinical trials (RCTs) are regarded as a golden standard for investigating the efficacy of treatments. Nevertheless, risk of bias (RoB) can still affect the validity of these trials. We aimed to conduct a systemic review of all control trials (CTs) using regenerative materials for alveolar cleft reconstructions to evaluate their RoB and perform a meta-analysis of new bone formation. METHODS: Cochrane Oral Health Group's Trials Register, the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE (PubMed), EMBASE AND Google Scholar were searched up to October 2020. Thereafter, the articles underwent quality assessment (according to the Jadad scale and the Delphi list) for the evaluation of the RoB. RESULTS: A total of 15 trials met the inclusion criteria, none of which reached a full score. Of these, 20% didn't randomize the trails, 73,33% failed to describe the way of randomization, and none reported the double-blinded criteria. Furthermore, allocation concealment (99.9%), intention to treat (100%), and patient awareness (100%) were inadequately described. The meta-analysis found no significant difference between regenerative materials and iliac crest graft. CONCLUSION: This review showed high RoB in CTs implying quality improvement of CTs is necessary. Meta-analysis showed no significant difference between the regenerative materials and autogenous grafts.
Assuntos
Enxerto de Osso Alveolar , Fissura Palatina , Autoenxertos , Fissura Palatina/cirurgia , Humanos , ÍlioRESUMO
There has been an increasing trend in oral squamous cell carcinoma (OSCC) in patients under 45 years of age. The aim of this study was to evaluate the burden of OSCC in the Netherlands between 1989 and 2018 among young adults (age 20-34 years) when compared to adults (age 35-44 years), and to describe the burden in older groups as well, utilizing cancer registry data to characterize incidence patterns by age, sex, and risk factors. A total of 18,963 cases of OSCC were reported. The overall incidence rate, as measured by annual percentage change (APC), increased significantly from 1989 to 2010 by 1.3% per year (95% confidence interval (CI) 0.9-1.7%) but decreased thereafter by -0.9% (95% CI -2.5% to 0.7%). Annual incidence increased significantly by 2.4% (95% CI 1.1-3.8%) for patients aged 20-34 years, while it decreased for those aged 35-44 years by -0.9% (95% CI -1.7% to 0.0%). In patients older than 60 years, incidence rates increased overall (60-74 years: APC 1.8%, 95% CI 1.5-2.1%; ≥75 years: APC 1.5%, 95% CI 1.2-1.9%). Overall, 66.5% of patients were smokers and 65.3% were alcohol consumers. The marked differences in incidence within the young age subgroups warrants further investigation to elucidate any likely disparity in biological process and clinical outcomes in these populations.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Adulto , Idoso , Humanos , Incidência , Países Baixos , Sistema de Registros , Carcinoma de Células Escamosas de Cabeça e Pescoço , Adulto JovemRESUMO
OBJECTIVE: Bone grafting is an important surgical procedure to restore missing bone in patients with alveolar cleft lip/palate, aiming to stabilize either sides of the maxillary segments by inducing new bone formation, and in bilateral cleft cases also to stabilize the pre-maxilla. Polyphosphate (PolyP), a physiological polymer composed of orthophosphate units linked together with high-energy phosphate bonds, is a naturally existing compound in platelets which, when complexed with calcium as Ca-polyP microparticles (Ca-polyP MPs), was proven to have osteoinductive properties in preclinical studies. AIM: To evaluate the feasibility, safety, and osteoinductivity of Ca-polyP MPs as a bone-inducing graft material in humans. METHODS: This prospective non-blinded first-in-man clinical pilot study shall consist of 8 alveolar cleft patients of 13 years or older to evaluate the feasibility and safety of Ca-PolyP MPs as a bone-inducing graft material. Patients will receive Ca-polyP graft material only or Ca-polyP in combination with biphasic calcium phosphate (BCP) as a bone substitute carrier. During the trial, the participants will be investigated closely for safety parameters using radiographic imaging, regular blood tests, and physical examinations. After 6 months, a hollow drill will be used to prepare the implantation site to obtain a biopsy. The radiographic imaging will be used for clinical evaluation; the biopsy will be processed for histological/histomorphometric evaluation of bone formation. DISCUSSION: This is the first-in-man study evaluating the safety and feasibility of the polyP as well as the potential regenerative capacity of polyP using an alveolar cleft model. TRIAL REGISTRATION: Indonesian Trial Registry INA-EW74C1N . Registered on 12 June 2020.
Assuntos
Fenda Labial , Fissura Palatina , Fenda Labial/diagnóstico por imagem , Fenda Labial/cirurgia , Fissura Palatina/diagnóstico por imagem , Fissura Palatina/cirurgia , Humanos , Indonésia , Projetos Piloto , Polifosfatos/efeitos adversos , Estudos Prospectivos , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
The aim of this study was to design and manufacture an easily assembled cartilage implant model for auricular reconstruction. First, the printing accuracy and mechanical properties of 3D-printed poly-ε-caprolactone (PCL) scaffolds with varying porosities were determined to assess overall material properties. Next, the applicability of alginate as cell carrier for the cartilage implant model was determined. Using the optimal outcomes of both experiments (in terms of (bio)mechanical properties, cell survival, neocartilage formation, and printing accuracy), a hybrid auricular implant model was developed. PCL scaffolds with 600 µm distances between strands exhibited the best mechanical properties and most optimal printing quality for further exploration. In alginate, chondrocytes displayed high cell survival (~83% after 21 days) and produced cartilage-like matrix in vitro. Alginate beads cultured in proliferation medium exhibited slightly higher compressive moduli (6 kPa) compared to beads cultured in chondrogenic medium (3.5 kPa, p > .05). The final auricular mold could be printed with 300 µm pores and high fidelity, and the injected chondrocytes survived the culture period of 21 days. The presented hybrid auricular mold appears to be an adequate model for cartilage tissue engineering and may provide a novel approach to auricular cartilage regeneration for facial reconstruction. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1711-1721, 2019.
Assuntos
Alginatos/química , Materiais Biocompatíveis/química , Cartilagem da Orelha/metabolismo , Hidrogéis/química , Poliésteres/química , Alicerces Teciduais/química , Animais , Materiais Biocompatíveis/metabolismo , Fenômenos Biomecânicos , Bioprótese , Proliferação de Células/efeitos dos fármacos , Condrócitos/citologia , Condrogênese/efeitos dos fármacos , Cabras , Hidrogéis/metabolismo , Poliésteres/metabolismo , Porosidade , Impressão Tridimensional , Regeneração , Propriedades de Superfície , Engenharia TecidualRESUMO
OBJECTIVES: Studies which consider the molecular mechanisms of degeneration and regeneration of cartilaginous tissues are seriously hampered by problematic ribonucleic acid (RNA) isolations due to low cell density and the dense, proteoglycan-rich extracellular matrix of cartilage. Proteoglycans tend to co-purify with RNA, they can absorb the full spectrum of UV light and they are potent inhibitors of polymerase chain reaction (PCR). Therefore, the objective of the present study is to compare and optimise different homogenisation methods and RNA isolation kits for an array of cartilaginous tissues. MATERIALS AND METHODS: Tissue samples such as the nucleus pulposus (NP), annulus fibrosus (AF), articular cartilage (AC) and meniscus, were collected from goats and homogenised by either the MagNA Lyser or Freezer Mill. RNA of duplicate samples was subsequently isolated by either TRIzol (benchmark), or the RNeasy Lipid Tissue, RNeasy Fibrous Tissue, or Aurum Total RNA Fatty and Fibrous Tissue kits. RNA yield, purity, and integrity were determined and gene expression levels of type II collagen and aggrecan were measured by real-time PCR. RESULTS: No differences between the two homogenisation methods were found. RNA isolation using the RNeasy Fibrous and Lipid kits resulted in the purest RNA (A260/A280 ratio), whereas TRIzol isolations resulted in RNA that is not as pure, and show a larger difference in gene expression of duplicate samples compared with both RNeasy kits. The Aurum kit showed low reproducibility. CONCLUSION: For the extraction of high-quality RNA from cartilaginous structures, we suggest homogenisation of the samples by the MagNA Lyser. For AC, NP and AF we recommend the RNeasy Fibrous kit, whereas for the meniscus the RNeasy Lipid kit is advised.Cite this article: M. Peeters, C. L. Huang, L. A. Vonk, Z. F. Lu, R. A. Bank, M. N. Helder, B. Zandieh Doulabi. Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis. Bone Joint Res 2016;5:560-568. DOI: 10.1302/2046-3758.511.BJR-2016-0033.R3.
RESUMO
Successful stem cell therapy after acute myocardial infarction (AMI) is hindered by lack of engraftment of sufficient stem cells at the site of injury. We designed a novel technique to overcome this problem by assembling stem cell-microbubble complexes, named 'StemBells'. StemBells were assembled through binding of dual-targeted microbubbles (~3µm) to adipose-derived stem cells (ASCs) via a CD90 antibody. StemBells were targeted to the infarct area via an ICAM-1 antibody on the microbubbles. StemBells were characterized microscopically and by flow cytometry. The effect of ultrasound on directing StemBells towards the vessel wall was demonstrated in an in vitro flow model. In a rat AMI-reperfusion model, StemBells or ASCs were injected one week post-infarction. A pilot study demonstrated feasibility of intravenous StemBell injection, resulting in localization in ICAM-1-positive infarct area three hours post-injection. In a functional study five weeks after injection of StemBells cardiac function was significantly improved compared with controls, as monitored by 2D-echocardiography. This functional improvement neither coincided with a reduction in infarct size as determined by histochemical analysis, nor with a change in anti- and pro-inflammatory macrophages. In conclusion, the StemBell technique is a novel and feasible method, able to improve cardiac function post-AMI in rats.
Assuntos
Microbolhas , Infarto do Miocárdio/terapia , Transplante de Células-Tronco/métodos , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Administração Intravenosa , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Ecocardiografia , Coração/diagnóstico por imagem , Coração/fisiopatologia , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Projetos Piloto , Ratos , Ratos Wistar , Sonicação , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
INTRODUCTION: Ear reconstruction is a tedious and demanding surgical procedure and the implant framework used is essential for the esthetic result. The outcome of a reconstructed ear, however, is not necessarily limited to the implant shape but rather to the available options of transplantable tissue for coverage. Apart from the visual aesthetics, ear reconstruction subsequently also requires implant dimensions to be adapted to the surgical possibilities. In this article, we have brought different disciplines together to develop a customizable ear model for 3D printing of ear implants. MATERIAL AND METHODS: Computed tomography (CT) scans were made of 4 human cadaver ears before and after soft tissue dissection using a Discovery 750 High Definition Freedom Edition scanner (GE, Milwaukee, WI, USA) and subsequently converted into an STL data set using Mimics Software (Materialise, Leuven, Belgium). These scans were then used to develop a fully adjustable parametric model based on the essential ear anatomy using Rhinoceros and Grasshopper software. RESULTS: To determine the quality of the developed models, directed Hausdorff distance (DHD) was applied as the basis for measuring the similarity between the parametric model and the ear cartilage scanning data. Two methods were used. The mean directed Haussdorff distance (MDHD) was calculated based on the distribution of point sets showing an average similarity of 0.8 mm (±0.05 mm). The mean similarity coefficient (SC) of the model and scan surfaces was 94% with a 2-mm threshold. CONCLUSION: This study shows that a parametric standard model could be used as a feasible method to generate custom implants based on existing ear images.
Assuntos
Orelha Externa/anatomia & histologia , Modelagem Computacional Específica para o Paciente , Procedimentos de Cirurgia Plástica/métodos , Algoritmos , Cadáver , Desenho Assistido por Computador , Orelha Externa/cirurgia , Estudos de Viabilidade , Humanos , Processamento de Imagem Assistida por Computador/métodos , Impressão Tridimensional , Próteses e Implantes , Desenho de Prótese , Tomografia Computadorizada por Raios X/métodosRESUMO
PURPOSE: To relate the progress of vertebral segmental stability after interbody fusion surgery with radiological assessment of spinal fusion. METHODS: Twenty goats received double-level interbody fusion and were followed for a period of 3, 6 and 12 months. After killing, interbody fusion was assessed radiographically by two independent observers. Subsequently, the lumbar spines were subjected to four-point bending and rotational deformation, assessed with an optoelectronic 3D movement registration system. In addition, four caprine lumbar spines were analysed in both the native situation and after the insertion of a cage device, as to mimic the direct post-surgical situation. The range of motion (ROM) in flexion/extension, lateral bending and axial rotation was analysed ex vivo using a multi-segment testing system. RESULTS: Significant reduction in ROM in the operated segments was already achieved with moderate bone ingrowth in flexion/extension (71 % reduction in ROM) and with only limited bone ingrowth in lateral bending (71 % reduction in ROM) compared to the post-surgical situation. The presence of a sentinel sign always resulted in a stable vertebral segment in both flexion/extension and lateral bending. For axial rotation, the ROM was already limited in both native and cage inserted situations, resulting in non-significant differences for all radiographic scores. DISCUSSION: In vivo vertebral segment stability, defined as a significant reduction in ROM, is achieved in an early stage of spinal fusion, well before a radiological bony fusion between the vertebrae can be observed. Therefore, plain radiography underestimates vertebral segment stability.
Assuntos
Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/cirurgia , Modelos Animais , Fusão Vertebral/métodos , Animais , Fenômenos Biomecânicos , Feminino , Seguimentos , Cabras , Vértebras Lombares/fisiopatologia , Movimento , Radiografia , Amplitude de Movimento Articular , Rotação , Fusão Vertebral/instrumentação , Suporte de Carga/fisiologiaRESUMO
BACKGROUND: Osteosarcoma (OS) is the most common bone tumour in children and adolescents. Despite aggressive therapy regimens, treatment outcomes are unsatisfactory. Targeted delivery of drugs can provide higher effective doses at the site of the tumour, ultimately improving the efficacy of existing therapy. Identification of suitable receptors for drug targeting is an essential step in the design of targeted therapy for OS. METHODS: We conducted a comparative analysis of the surface proteome of human OS cells and osteoblasts using cell surface biotinylation combined with nano-liquid chromatography - tandem mass spectrometry-based proteomics to identify surface proteins specifically upregulated on OS cells. This approach generated an extensive data set from which we selected a candidate to study for its suitability as receptor for targeted treatment delivery to OS. First, surface expression of the ephrin type-A receptor 2 (EPHA2) receptor was confirmed using FACS analysis. Ephrin type-A receptor 2 expression in human tumour tissue was tested using immunohistochemistry. Receptor targeting and internalisation studies were conducted to assess intracellular uptake of targeted modalities via EPHA2. Finally, tissue micro arrays containing cores of human OS tissue were stained using immunohistochemistry and EPHA2 staining was correlated to clinical outcome measures. RESULTS: Using mass spectrometry, a total of 2841 proteins were identified of which 156 were surface proteins significantly upregulated on OS cells compared with human primary osteoblasts. Ephrin type-A receptor 2 was highly upregulated and the most abundant surface protein on OS cells. In addition, EPHA2 was expressed in a vast majority of human OS samples. Ephrin type-A receptor 2 effectively mediates internalisation of targeted adenoviral vectors into OS cells. Patients with EPHA2-positive tumours showed a trend toward inferior overall survival. CONCLUSION: The results presented here suggest that the EPHA2 receptor can be considered an attractive candidate receptor for targeted delivery of therapeutics to OS.
Assuntos
Neoplasias Ósseas/metabolismo , Osteossarcoma/metabolismo , Receptor EphA2/análise , Receptor EphA2/metabolismo , Neoplasias Ósseas/química , Neoplasias Ósseas/tratamento farmacológico , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Mineração de Dados , Feminino , Citometria de Fluxo/métodos , Humanos , Masculino , Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Osteossarcoma/química , Osteossarcoma/tratamento farmacológico , Prognóstico , Proteoma/análise , Proteoma/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Regulação para CimaRESUMO
The secretome of stem cells strongly determines the outcome of tissue engineering strategies. We investigated how the secretome of human adipose stem cells (hASCs) can be affected by substrate, BMP-2 treatment, and degree of differentiation. We hypothesized that as differentiation progresses, hASCs produce increasingly more gene products associated with processes such as angiogenesis and bone remodeling. Human ASCs were treated for 15 min with BMP-2 (10 ng/ml) to enhance osteogenic differentiation, or with vehicle. Subsequently, hASCs were seeded on plastic or on biphasic calcium phosphate (BCP) consisting of 60% hydroxyapatite and 40% ß-tricalcium phosphate. A PCR array for ~150 trophic factors and differentiation-related genes was performed at day 21 of culture. A limited set of factors was quantified by qPCR at days 0, 4, 14 and 21, and/or ELISA at day 21. Compared to plastic, BCP-cultured hASCs showed ≥2-fold higher expression of ~20 factors, e.g. cytokines such as IL-6, growth factors such as FGF7 and adhesion molecules such as VCAM1. Expression of another ~50 genes was decreased ≥2-fold on BCP vs. plastic, even though hASCs differentiate better on BCP than on plastic. BMP-2-treatment increased the expression of ~30 factors by hASCs seeded on BCP, while it decreased the expression of only PGF, PPARG and PTN. Substrate affected hASC secretion of Activin A and seemed to affect P1NP release. No clear association between hASC osteogenic differentiation and growth factor expression pattern was observed. Considering our observed lack of association between the degree of differentiation and the expression of factors associated with angiogenesis and bone remodeling by hASCs, future bone regeneration studies should focus more on systematically orchestrating the secretome of stem cells, rather than on inducing osteogenic differentiation of stem cells only. Short incubation with BMP-2 may be a promising treatment to enhance both osteogenic differentiation and environmental modulation.
Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Células-Tronco/metabolismo , Transcriptoma/fisiologia , Tecido Adiposo/citologia , Diferenciação Celular , Células Cultivadas , Humanos , Hidroxiapatitas , Osteogênese , Células-Tronco/efeitos dos fármacos , Engenharia TecidualRESUMO
Adipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically, transmission of pathogens and antibody development against FBS are possible. In this study, we investigated whether FBS can be substituted by human platelet lysate (PL) in ASC culture, without affecting functional capacities particularly important for cardiac repair application of ASC. We found that PL-cultured ASC had a significant 3-fold increased proliferation rate and a significantly higher attachment to tissue culture plastic as well as to endothelial cells compared with FBS-cultured ASC. PL-cultured ASC remained a significant 25% smaller than FBS-cultured ASC. Both showed a comparable surface marker profile, with the exception of significantly higher levels of CD73, CD90, and CD166 on PL-cultured ASC. PL-cultured ASC showed a significantly higher migration rate compared with FBS-cultured ASC in a transwell assay. Finally, FBS- and PL-cultured ASC had a similar high capacity to differentiate towards cardiomyocytes. In conclusion, this study showed that culturing ASC is more favorable in PL-supplemented medium compared with FBS-supplemented medium.
Assuntos
Tecido Adiposo/citologia , Plaquetas/metabolismo , Substitutos Sanguíneos/farmacologia , Extratos Celulares/farmacologia , Miocárdio/patologia , Soro/metabolismo , Cicatrização/efeitos dos fármacos , Adulto , Idoso , Animais , Biomarcadores/metabolismo , Plaquetas/efeitos dos fármacos , Bovinos , Adesão Celular/efeitos dos fármacos , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Citometria de Fluxo , Humanos , Pessoa de Meia-Idade , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismoRESUMO
Gonadotrophin surge-inhibiting/attenuating factor (GnSIF/AF) has been known for over two decades, but its molecular structure has not been completely characterized yet. In the last 20 years, five different putative GnSIF/AF sequences have been published. In this article, we describe a procedure to isolate and characterize GnSIF/AF from bovine follicular fluid, a GnSIF/AF-derived synthetic peptide (SP-GnSIF/AF) was produced, and the intracellular bioactivity of GnSIF/AF was tested for intracellular action with a MAPK-assay. Two different bioactive molecular weight forms of GnSIF/AF were isolated, a 160 kDa heteromeric and a monomeric 40 kDa protein. The 40 kDa form appeared to be a subunit of the 160 kDa protein. The synthetic peptide mimicked the actions of GnSIF/AF, such as inhibition of GnRH-induced LH secretion and attenuation of the MAPK phosphorylation. The two GnSIF/AF candidates do not show similarities with previously published GnSIF/AF sequences. These are the first data showing the influence of GnSIF/AF on intracellular processes involved in GnRH self-priming and that the biological action of GnSIF/AF was preserved in the produced synthetic peptide. The results provide strong evidence that the identified candidate proteins are the true GnSIF/AF.
Assuntos
Hormônios Gonadais , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Hormônio Liberador de Gonadotropina/análise , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas , Animais , Bovinos , Feminino , Líquido Folicular/química , Hormônios Gonadais/síntese química , Hormônios Gonadais/isolamento & purificação , Hormônios Gonadais/fisiologia , Hormônio Luteinizante/análise , Hormônio Luteinizante/antagonistas & inibidores , Hormônio Luteinizante/metabolismo , Camundongos , Peso Molecular , Proteínas/síntese química , Proteínas/isolamento & purificação , Proteínas/fisiologia , Ratos , Ratos WistarRESUMO
Bioabsorbable polymers are increasingly being used in tissue engineering strategies. Despite the knowledge that some sterilization techniques may affect the physical properties of these polymers, this aspect is often overlooked. We speculate that the type of sterilization method used may influence cellular responses by altering the surface characteristics. We cultured adipose stem cells on bioabsorbable poly(l-lactide-co-caprolactone) (PLCL) sheets, sterilized using either ethylene oxide (EO), argon glow discharge (aGD) or electron beam (e-beam). Significantly higher values for surface roughness in the order EO>aGD>e-beam and significant differences in contact angles (EO>e-beam>aGD) and surface energies (aGD>e-beam>EO) were observed. Increased cell attachment and proliferation rates were observed with lower contact angles. The alkaline phosphatase activity was significantly higher for the ethylene oxide sterilized PLCL sheet. In conclusion, the type of sterilization for bioabsorbable polymers should be considered in the design of new scaffolds, since it might affect, or can be used to enhance, the outcome of the tissue engineered construct.
Assuntos
Adipócitos/citologia , Substitutos Ósseos/síntese química , Óxido de Etileno/química , Osteoblastos/citologia , Poliésteres/química , Células-Tronco/citologia , Engenharia Tecidual/métodos , Adipócitos/fisiologia , Diferenciação Celular , Células Cultivadas , Elétrons , Gases/química , Temperatura Alta , Humanos , Teste de Materiais , Osteoblastos/fisiologia , Células-Tronco/fisiologia , Propriedades de SuperfícieRESUMO
Beta1 integrins play a controversial role during chondrogenesis. Since the maturation of chondrocytes relies on a signaling switch from cell-cell to cell-matrix interactions, we hypothesized that beta1 integrins play a different role at the earlier (mainly cell-cell interaction) from the later stage (mainly cell-matrix interaction) of chondrogenesis. Our data showed: in plain medium, sox9, collagen X, and collagen II gene expressions of ASCs were induced by beta1-integrin blockage at day 14. In chondrogenic medium, however, sox 9, sox6, and collagen II gene expression were induced at day 4 but inhibited at day 14. In addition, both beta1-integrin blockage and TGF-beta1 down-regulated Rock-1 and -2 gene expression and produced the round cells. We concluded that beta1 integrins play a more important role at the later stages than earlier stages of chondrogenesis, and that the onset of chondrogenesis promoted by beta1-integrin blockage might be through inhibiting Rock signaling.
Assuntos
Tecido Adiposo/citologia , Condrogênese , Regulação da Expressão Gênica no Desenvolvimento , Integrina beta1/fisiologia , Células-Tronco/citologia , Quinases Associadas a rho/genética , Tecido Adiposo/efeitos dos fármacos , Forma Celular/genética , Condrogênese/genética , Colágeno Tipo II/genética , Colágeno Tipo X/genética , Meios de Cultura , Proteínas de Ligação a DNA/genética , Expressão Gênica/efeitos dos fármacos , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Imunoglobulina G/farmacologia , Integrina beta1/farmacologia , Fatores de Transcrição SOX9 , Fatores de Transcrição SOXD , Células-Tronco/efeitos dos fármacos , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/farmacologia , Fator de Crescimento Transformador beta1/fisiologiaRESUMO
New regenerative treatment strategies are being developed for intervertebral disc degeneration of which the implantation of various cell types is promising. All cell types used so far require in vitro expansion prior to clinical use, as these cells are only limited available. Adipose-tissue is an abundant, expendable and easily accessible source of mesenchymal stem cells. The use of these cells therefore eliminates the need for in vitro expansion and subsequently one-step regenerative treatment strategies can be developed. Our group envisioned, described and evaluated such a one-step procedure for spinal fusion in the goat model. In this review, we summarize the current status of cell-based treatments for intervertebral disc degeneration and identify the additional research needed before adipose-derived mesenchymal stem cells can be evaluated in a one-step procedure for regenerative treatment of the intervertebral disc. We address the selection of stem cells from the stromal vascular fraction, the specific triggers needed for cell differentiation and potential suitable scaffolds. Although many factors need to be studied in more detail, potential application of a one-step procedure for intervertebral disc regeneration seems realistic.
Assuntos
Adipócitos/transplante , Disco Intervertebral , Transplante de Células-Tronco Mesenquimais/tendências , Doenças da Coluna Vertebral/terapia , Animais , Humanos , Disco Intervertebral/patologia , Disco Intervertebral/fisiopatologia , Regeneração , Doenças da Coluna Vertebral/patologia , Doenças da Coluna Vertebral/fisiopatologia , Fusão Vertebral/tendências , Engenharia Tecidual/tendênciasRESUMO
Tissue microenvironment plays a critical role in guiding local stem cell differentiation. Within the intervertebral disc, collagen type II and nucleus pulposus (NP) cells are two major components. This study aimed to investigate how collagen type II and NP cells affect adipose tissue-derived stem cells (ASCs) in a 3D environment. ASCs were cultured in collagen type I or type II hydrogels alone, or co-cultured in transwells with micromass NP cells for 4 and 14 days. ASCs seeded in collagen type II gels acquired dentritic cell shapes, and orchestrated cell density-dependent gel contraction rates. Up-regulation of collagen type X, but not of other chondrogenic markers was observed at day 4, irrespective of the hydrogel type. Strikingly, in co-cultures with NP cells, more pronounced differentiation of ASCs along the cartilaginous lineage was observed (up-regulation of collagen IIA, IIB and aggrecan gene expression, as well as stronger alcian blue staining), when ASCs were embedded in collagen type II in comparison with type I hydrogels. Interestingly, strong cellular condensations/aggregations were observed in ASC-seeded type II, but not type I gels, and this aggregation was markedly delayed when the same gels were co-cultured with NP cells. The NP cell-mediated inhibition of ASC aggregation in collagen type II gels coincided with down-regulation of integrin subunit alpha2 gene expression. We conclude that soluble factors released by NP cells can direct chondrogenic differentiation of ASCs in collagen hydrogels, and that combination with a nucleus-mimicking collagen type II microenvironment enhances differentiation towards a more pronounced cartilage/NP lineage relative to collagen type I hydrogels.
Assuntos
Tecido Adiposo/citologia , Diferenciação Celular/efeitos dos fármacos , Colágeno Tipo II/farmacologia , Disco Intervertebral/citologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Adolescente , Adulto , Cartilagem/citologia , Cartilagem/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Contagem de Células , Linhagem da Célula/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Regulação para Baixo/efeitos dos fármacos , Feminino , Géis , Humanos , Integrina alfa2/genética , Integrina alfa2/metabolismo , Masculino , Pessoa de Meia-Idade , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Células-Tronco/metabolismoRESUMO
For bone tissue engineering, it is important that mesenchymal stem cells (MSCs) differentiate into osteoblasts. To develop a method for differentiation of adipose tissue-derived mesenchymal stem cells (AT-MSCs) along the osteogenic lineage, we studied the effect of polyamines, which are organic cations implicated in bone growth and development, on differentiation of AT-MSCs. Treatment of goat-derived AT-MSCs with 1,25-dihydroxyvitamin-D3 (1,25(OH)(2)D(3)), which stimulates osteogenic differentiation, for 7 days induced gene expression of the polyamine-modulated transcription factor-1 (PMF-1) and spermidine/spermine N (1)-acetyltransferase (SSAT), which are both involved in polyamine metabolism, suggesting that polyamines are involved in osteogenic differentiation of AT-MSCs. Furthermore, treatment of AT-MSCs with the polyamine spermine-regulated gene expression of runx-2, a transcription factor involved in early stages of osteogenic differentiation, and that of osteopontin, a bone matrix protein expressed in later stages of osteogenic differentiation. Runx-2 gene expression was increased 4 and 14 days after a short 30 min. treatment with spermine, while osteopontin gene expression was only increased 4 days after spermine treatment. Finally, alkaline phosphatase activity, which is intimately involved in the formation of extracellular matrix of bone, was increased 4 weeks after the 30 min.-spermine treatment of AT-MSCs. In conclusion, this study shows for the first time that the polyamine spermine regulates differentiation of AT-MSCs along the osteogenic lineage, which can be used as a new method for differentiation of AT-MSCs along the osteogenic lineage. Therefore, polyamines may constitute a promising tool for bone tissue engineering approaches using AT-MSCs, such as a one-step surgical procedure for spinal interbody fusion.
Assuntos
Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Espermina/farmacologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Acetiltransferases/genética , Acetiltransferases/metabolismo , Animais , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Cabras , Osteopontina/genética , Osteopontina/metabolismoRESUMO
Adipose tissue-derived mesenchymal stem cells (AT-MSCs) are currently used for bone tissue engineering. AT-MSCs undergoing osteogenic differentiation respond to mechanical loading with increased cyclooxygenase-2 gene expression, a key enzyme in prostaglandin (PG) synthesis. PGs are potent multifunctional regulators in bone, exhibiting stimulatory and inhibitory effects on bone formation and resorption. PGE(2), but not PGI(2) or PGF(2), recruits osteoprogenitors from the bone marrow space and influences their differentiation. We hypothesize that PGE(2), PGI(2), and PGF(2) may differentially regulate osteogenic differentiation of human AT-MSCs. PGE(2), PGI(2), and PGF(2) (0.01-10 microM) affected osteogenic differentiation, but not proliferation of AT-MSCs after 4-14 days. Only PGF(2) (0.01-10 microM) increased alkaline phosphatase (ALP) activity at day 4. PGE(2) (10 microM), PGI(2) (0.01-10 microM), and PGF(2) (10 microM) decreased ALP activity, whereas PGF(2) (0.1 microM) increased ALP activity at day 14. PGF(2) (0.01-0.1 microM) and PGI(2) (0.01 microM) upregulated osteopontin gene expression, and PGF(2) (0.01 microM) upregulated alpha1(I)procollagen gene expression at day 4. PGE(2) and PGF(2) (10 microM) at day 4 and PGF(2) (1 microM) at day 14 downregulated runt-related transcription factor-2 gene expression. We conclude that PGE(2), PGI(2), and PGF(2) differentially affect osteogenic differentiation of AT-MSCs, with PGF(2) being the most potent. Thus, locally produced PGF(2) might be most beneficial in promoting osteogenic differentiation of AT-MSCs, resulting in enhanced bone formation for bone tissue engineering.
Assuntos
Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Prostaglandinas/administração & dosagem , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Engenharia Tecidual/métodosRESUMO
Degenerative disc disease (DDD) is a major cause of chronic low back pain. For mild/intermediate DDD, regeneration by injecting adipose stem cells (ASCs) into the nucleus pulposus (NP) may be considered. The goal of this study is to investigate whether NP cells can direct ASCs towards the NP phenotype. Interactions between NP cells and ASCs were studied in transwell co-cultures, employing both monolayer and micromass configurations. Micromass culturing significantly up-regulated aggrecan and collagen type II gene expression in NP cells. In ASCs, expression of these genes and of osteopontin, collagen type I and PPAR-gamma were not significantly affected. Strikingly, only when both cell types were micromass-cultured, ASCs could be chondrogenically differentiated, as shown by induction of collagen type II and aggrecan, and concomitant down-regulation of osteopontin, collagen type I and PPAR-gamma. We conclude that ASCs can be directed towards the NP cell-like phenotype by soluble factor(s) secreted by NP cells.