RESUMEN
The use of adult mesenchymal stem cells (MSC) in cartilage tissue engineering has been implemented in the field of regenerative medicine and offers new perspectives in the generation of transplants for reconstructive surgery. The extracellular matrix (ECM) plays a key role in modulating function and phenotype of the embedded cells and contains the integrins as adhesion receptors mediating cell-cell and cell-matrix interactions. In our study, characteristic changes in integrin expression during the course of chondrogenic differentiation of MSC from bone marrow and foetal cord blood were compared. MSC were isolated from bone marrow biopsies and cord blood. During cell culture, chondrogenic differentiation was performed. The expression of integrins and their signalling components were analysed with microarray and immunohistochemistry in freshly isolated MSC and after chondrogenic differentiation. The fibronectin-receptor (integrin a5b1) was expressed by undifferentiated MSC, expression rose during chondrogenic differentiation in both types of MSC. The components of the vitronectin/osteopontin-receptors (avb5) were not expressed by freshly isolated MSC, expression rose with ongoing differentiation. Receptors for collagens (a1b1, a2b1, a3b1) were weakly expressed by undifferentiated MSC and were activated during differentiation. As intracellular signalling components integrin linked kinase (ILK) and CD47 showed increasing expression with ongoing differentiation. For all integrins, no significant differences could be found in the two types of MSC. Integrin-mediated signalling seems to play an important role in the generation and maintenance of the chondrocytic phenotype during chondrogenic differentiation. Especially the receptors for fibronectin, vitronectin, osteopontin and collagens might be involved in the generation of the ECM. Intracellularly, their signals might be transduced by ILK and CD47. To fully harness the potential of these cells, future studies should be directed to ascertain their cellular and molecular characteristics for optimal identification, isolation and expansion.
RESUMEN
Skeletal muscle tissue engineering, a promising specialty, aims at the reconstruction of skeletal muscle loss. In vitro tissue engineering attempts to achieve this goal by creating differentiated, functional muscle tissue through a process in which stem cells are extracted from the patient, e.g. by muscle biopsies, expanded and differentiated in a controlled environment, and subsequently re-implanted. A prerequisite for this undertaking is the ability to cultivate and differentiate human skeletal muscle cell cultures. Evidently, optimal culture conditions must be investigated for later clinical utilization. We therefore analysed the proliferation of human cells in different environments and evaluated the differentiation potential of different culture media. It was shown that human myoblasts have a higher rate of proliferation in the alamarBlue assay when cultured on gelatin-coated culture flasks rather than polystyrene-coated flasks. We also demonstrated that myoblasts treated with a culture medium with a high concentration of growth factors [growth medium (GM)] showed a higher proliferation compared to cultures treated with a culture medium with lower amounts of growth factors [differentiation medium (DM)]. Differentiation of human myoblast cell cultures treated with GM and DM was analysed until day 16 and myogenesis was verified by expression of MyoD, myogenin, alpha-sarcomeric actin and myosin heavy chain by semi-quantitative RT-PCR. Immunohistochemical staining for desmin, Myf-5 and alpha-sarcomeric actin was performed to verify the myogenic phenotype of extracted satellite cells and to prove the maturation of cells. Cultures treated with DM showed positive staining for alpha-sarcomeric actin. Notably, markers of differentiation were also detected in cultures treated with GM, but there was no formation of myotubes. In the enzymatic assay of creatine phosphokinase, cultures treated with DM showed a higher activity, evidencing a higher degree of differentiation. In this study, we obtained detailed information regarding the cultivation and differentiation of human myoblast cultures in different environments. By exploring optimal culture conditions for skeletal muscle tissue engineering, we acquired culture data for comparison with other sources of stem cells in order to find the most applicable stem cell for focussed clinical usage.
Asunto(s)
Mioblastos/citología , Ingeniería de Tejidos/métodos , Adulto , Anciano , Proliferación Celular , Células Cultivadas , Creatina Quinasa/metabolismo , Medios de Cultivo , Densitometría , Gelatina/metabolismo , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Proteínas Musculares/metabolismo , Mioblastos/enzimología , Oxazinas , Poliestirenos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , XantenosRESUMEN
The use of adult mesenchymal stem cells (MSC) in cartilage tissue engineering offers new perspectives in the generation of transplants for reconstructive surgery. The extracelular matrix (ECM) plays a key role in modulating the function and phenotype of the embedded cells and contains the integrins as adhesion receptors mediating cell-cell and cell-matrix interactions. In our study, characteristic changes in integrin expression during the course of chondrogenic differentiation of MSC from bone marrow and adipose tissue were compared. MSC were isolated from bone marrow biopsies and adipose tissue. During cell culture, chondrogenic differentiation was performed. The expression of integrins and their signaling components were analysed with microarray and immunohistochemistry in freshly isolated MSC and after chondrogenic differentiation. The fibronectin receptor (integrin alpha5beta1) was expressed by undifferentiated MSC, and expression rose during chondrogenic differentiation in both types of MSC. The components of the vitronectin/osteopontin receptors (alphavbeta5) were not expressed by freshly isolated MSC, and expression rose with ongoing differentiation. Receptors for the collagens (alpha1beta1, alpha2beta1, alpha3beta1) were weakly expressed by undifferentiated MSC and were activated during differentiation. Intracellular signaling components integrin-linked kinase (ILK) and CD47 showed increased expression with ongoing differentiation. For all integrins, no significant differences were be found in the 2 types of MSC. Integrin-mediated signaling appeared to play an important role in the generation and maintenance of the chondrocytic phenotype during chondrogenic differentiation. Particularly, the receptors for fibronectin, vitronectin, osteopontin and the collagens may be involved in the generation of the ECM. Intracellularly, their signals might be transduced by ILK and CD47. To fully harness the potential of these cells, future studies should be directed to ascertain their cellular and molecular characteristics for optimal identification, isolation, and expansion.
Asunto(s)
Tejido Adiposo/citología , Células de la Médula Ósea/citología , Diferenciación Celular , Condrogénesis , Integrinas/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Tejido Adiposo/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Células de la Médula Ósea/metabolismo , Separación Celular , Células Cultivadas , Femenino , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Integrinas/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Programas InformáticosRESUMEN
Skeletal muscle tissue engineering is a promising interdisciplinary specialty which aims at the reconstruction of skeletal muscle loss caused by traumatic injury congenital defects or tumor ablations. Due to the difficulty in procuring donor tissue, the possibilities for alternative treatment like autologous grafting (e.g. muscle flaps) are limited. This process also presents consistent problems with donor-site morbidity. Skeletal muscle tissue engineering tries to overcome this problem by generating new, functional muscle tissue from autologous precursor cells (stem cells). Multiple stem cells from different sources can be utilized for restoration of differentiated skeletal muscle tissue using tissue engineering principles. After 15 years of intensive research in this emerging field, for the first time, solutions using different strategies (e.g. embryonic stem cells, arterio-venous (AV) loop models, etc.) are being presented to resolve problems like vascularisation of tissue engineered constructs. This article reviews recent findings in skeletal muscle tissue engineering and outlines its relevance to clinical applications in reconstructive surgery.
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Músculo Esquelético/citología , Ingeniería de Tejidos , Animales , HumanosRESUMEN
Craniofacial tissue loss due to congenital defects, disease or injury is a major clinical problem. The head and neck region is composed of several tissues. The most prevalent method of reconstruction is autologous grafting. Often, there is insufficient host tissue for adequate repair of the defect side, and extensive donor site morbidity may result from the secondary surgical procedure. The field of tissue engineering has the potential to create functional replacements for damaged or pathologic tissues.
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Neoplasias de Cabeza y Cuello/cirugía , Procedimientos de Cirugía Plástica/métodos , Ingeniería de Tejidos , Humanos , Recolección de Tejidos y Órganos , Trasplante AutólogoRESUMEN
CONCLUSION: The palatal implant method originally designed to reduce snoring can significantly reduce the apnea-hypopnea index (AHI) in some patients with mild to moderate obstructive sleep apnea (OSA) in a single office-based procedure. OBJECTIVE: An initial study designed to evaluate the short-term efficacy and safety of palatal implants as primary treatment for patients with mild to moderate OSA. MATERIALS AND METHODS: This was a prospective, non-randomized study of 16 previously untreated and undiagnosed patients with sleep apnea. The inclusion criteria were an AHI of 10-30/h and a body mass index (BMI) < or = 30. RESULTS: The mean AHI was reduced following implantation, from 16.1 to 11.8 (p<0.01). A reduction in AHI was achieved in 13 patients (81%). Ten of 16 patients had their AHI reduced to <10.0. Snoring intensity decreased from 8.3+/-1.8 to 4.7+/-2.5 on a visual analog scale (p<0.001) and daytime sleepiness dropped from 7.2+/-2.5 to 4.6+/-3.2 on the Epworth Sleepiness Scale (p<0.05). No significant adverse events were reported.
Asunto(s)
Procedimientos Quirúrgicos Mínimamente Invasivos , Paladar Blando/cirugía , Tereftalatos Polietilenos , Implantación de Prótesis , Apnea Obstructiva del Sueño/cirugía , Adulto , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Polisomnografía , Complicaciones Posoperatorias/diagnóstico , Estudios Prospectivos , Diseño de Prótesis , Apnea Obstructiva del Sueño/diagnóstico , Ronquido/prevención & controlRESUMEN
Tissue engineering represents a promising method for generating chondrogenic grafts for reconstructive surgery. In cultured chondrocytes, the dedifferentiation of cells seems unavoidable for multiplication. Stem cells, however, displaying unlimited self-renewal and the capacity to differentiate towards chondrocytes, might be usable after further characterization. As the interactions between the extracellular matrix and the cellular compartment can alter the cellular behaviour, we investigated the expression of integrins using microarray analysis during chondrogenic differentiation of human mesenchymal stem cells (MSC) in comparison with de-differentiating human chondrocytes (HC) harvested during septoplasty. During chondrogenic differentiation of MSC, the fibronectin-receptor (Integrin beta1alpha5), fibronectin and the GPIIb/IIIa-receptor were downregulated. The components of the vitronectin-receptor (Integrin alphavbeta3) and CD47 were constantly expressed and ILK was downregulated. Vitronectin and osteopontin were not expressed by the cells. In HC, Integrin beta1alpha5 in conjunction with the ligand fibronectin were upregulated during dedifferentiation, Integrin alphavbeta3 as well as the GBIIb/IIIa-receptor were activated on day 21 but neither vitronectin nor osteopontin were expressed by the cells. The integrins, beta2, beta4, beta6, beta8 and alpha2, alpha4, alpha6, alpha7, alpha11, were not expressed at any time. ILK, CD47, and ICAP were activated with ongoing dedifferentiation. In conclusion, a candidate for signal-transmission is the fibronectin receptor (integrin alpha5beta1) in conjunction with its ligand fibronectin. Other receptors, e.g. for vitronectin and osteopontin (alphavbeta3), or their ligands do not seem to be involved in signal transmission for dedifferentiation. The GPIIb/IIIa-receptor might assist the process of dedifferentiation. Intracellularly, ILK, ICAP1 and CD47 might be involved in the transduction of integrin-dependent signals.
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Diferenciación Celular , Condrocitos/citología , Condrocitos/metabolismo , Expresión Génica/genética , Integrinas/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Células Cultivadas , Matriz Extracelular/genética , Humanos , Integrinas/genética , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
OBJECTIVES: Tissue engineering represents a promising method for the construction of autologous chondrogenic grafts for reconstructive surgery. In cultured chondrocytes, the dedifferentiation and proliferation of the cells are critical factors that influence the generation of transplants. The aim of our study was to find and characterize markers for cell proliferation and dedifferentiation in cultured chondrocytes. STUDY DESIGN AND SETTING: Human chondrocytes were isolated from septal cartilage and held in primary cell culture. Cells were harvested after 1, 6, and 21 days. The differentiation of the cells was investigated with bright-field microscopy, the expression patterns of various proteins using immunohistochemistry, and the expression of distinct genes with the microarray technique. RESULTS: The chondrocytes showed a strong proliferation. After 6 and 21 days, collagen 9 and 10 were downregulated; collagen 11 was activated. Collagen 1 and 2 were downregulated after 6 days but were reactivated after 21 days. Tumor growth factor beta (TGF-beta)1 was strongly expressed on days 1, 6, and 21, TGF-beta2 was never expressed, and TGF-beta3 and -beta4 were upregulated from day 1 to day 21. The TGF-beta receptor III was expressed on days 1, 6, and 21. Integrin beta1, beta5, and alpha5 were upregulated from day 1 to day 21; integrin beta3 was downregulated. CONCLUSION AND SIGNIFICANCE: Collagens 3, 4, 8, 9, and 11 might be new markers for the dedifferentiation of chondrocytes. Collagen 2 might be a marker for the synthetic activity of the cells rather than the dedifferentiation. TGF-beta3 and -beta4 might influence the dedifferentiation, which is fortified by the expression of TGF-beta receptor III. Integrin beta1, beta5, and alpha5 might be involved in signal transmission for the dedifferentiation.
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Condrocitos/metabolismo , Colágeno/análisis , Sustancias de Crecimiento/análisis , Integrinas/análisis , Biomarcadores/análisis , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Colágeno Tipo I/análisis , Colágeno Tipo II/análisis , Colágeno Tipo IX/análisis , Colágeno Tipo X/análisis , Colágeno Tipo XI/análisis , Regulación hacia Abajo , Humanos , Integrina alfa5/análisis , Cadenas beta de Integrinas/análisis , Integrina beta1/análisis , Integrina beta3/análisis , Proteoglicanos/análisis , Receptores de Factores de Crecimiento Transformadores beta/análisis , Factor de Crecimiento Transformador beta/análisis , Factor de Crecimiento Transformador beta1 , Factor de Crecimiento Transformador beta3 , Regulación hacia ArribaRESUMEN
Tissue engineering is a multidisciplinary field combining biology and engineering along with clinical application to design, manufacture, modify, grow and maintain living tissue. This field has enjoyed tremendous growth in the past 10 years fueled by its potential role in regenerating new tissues and naturally healing injured or diseased organs. Many approaches to tissue engineering have been explored, including ex vivo de novo construction of tissues and strategies of in vivo induction of tissue regeneration. Interventions are hindered by factors such as rejection by the immune system, limited blood supply or morbidity of the donor site. Regardless of the approach, most researchers and clinicians agree that any successful tissue engineering construct will derive from a single unit, the cell. Because the engineering of tissue necessitates a sufficient number of tissue-specific cells with minimal donor site morbidity, a great deal of scientific effort has been directed towards stem cell research and the use of stem cells as a source of cells for new tissues. This review aims at outlining the role of stem cells in tissue engineering, focusing on the use of adult-derived stem cells as applied to the research and practice of plastic surgery.
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Órganos Bioartificiales , Procedimientos de Cirugía Plástica , Trasplante de Células Madre , Células Madre/citología , Ingeniería de Tejidos/métodos , Animales , Técnicas de Cultivo de Célula , HumanosRESUMEN
Cartilage tissue engineering plays an important role in the generation of grafts for reconstructive surgery. In cultured chondrocytes, the dedifferentiation of cells seems unavoidable for multiplication. Dedifferentiated cells produce matrix of less quality, and the molecular basis is still not well understood. Therefore, the aim of our study was to investigate the expression of matrix modulators in human chondrocytes during expansion. Human chondrocytes were isolated from septal cartilage (n=32) and held in primary cell culture. Cells were harvested after 1, 6 and 21 days. The differentiation of cells using light microscopy, the expression patterns of various proteins (MMPs, BMPs, and TIMPs) using immunohistochemistry, and the expression of distinct genes using microarray technique, were investigated. The chondrocytes showed strong in vitro proliferation. After 6 and 21 days, BMP-5 and -8 were up-regulated, BMP-2 was down-regulated and BMP-6 was inactivated. Other BMPs were not expressed. The expression of MMP-2, -3 and -13 was up-regulated from day 1 to 21, and MMP-12 and -20 were down-regulated. Other MMPs were not expressed. TIMP-1 was up-regulated and TIMP-3 was down-regulated during expansion. Differential expression of matrix modulators might influence the matrix composition of engineered cartilage. Improving the basic knowledge in this area may ultimately help clinicians to identify and proactively intervene in an attempt to prevent bioartificial cartilage from losing stability.
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Condrocitos/metabolismo , Matriz Extracelular/metabolismo , Perfilación de la Expresión Génica , Ingeniería de Tejidos/métodos , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 5 , Proteínas Morfogenéticas Óseas/análisis , Proteínas Morfogenéticas Óseas/genética , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Condrocitos/química , Condrocitos/citología , Matriz Extracelular/química , Humanos , Inmunohistoquímica , Metaloproteinasas de la Matriz/análisis , Metaloproteinasas de la Matriz/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de Tiempo , Inhibidores Tisulares de Metaloproteinasas/análisis , Inhibidores Tisulares de Metaloproteinasas/genética , Factor de Crecimiento Transformador beta/análisis , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Cartilage tissue engineering has an important role to play in the generation of graft material for reconstructive surgery. In cultured chondrocytes, the dedifferentiation of cells seems unavoidable for multiplication. Dedifferentiated cells produce matrix of less quality. Normal cartilage is composed of chondrocytes, which are embedded within an extracellular matrix (ECM). The ECM plays a key role in controlling cellular characteristics and contains the integrins as a large family of heterodimeric cell adhesion receptors involved in cell-cell and cell-matrix interactions. In this study, the characteristic changes of integrin expression and expression of matrix proteins during the course of dedifferentiation of chondrocytes in cell culture for 1, 6 and 21 days, analyzed at the mRNA level by microarray analysis and at the protein level by immunohistochemistry, are described. The components of the fibronectin receptor, integrin beta1,alpha5, in conjunction with the ligand fibronectin, were up-regulated during dedifferentiation. Integrin beta3 was expressed in the grey area. The components of the vitronectin-receptor, integrin alpha2b, alpha v, as well as integrin beta5, were activated on day 21, but neither vitronectin nor osteopontin were expressed by the cells. With ongoing dedifferentiation, activation of the GPIIb/GPIIIa receptor was found. The integrins beta2, beta4, beta6, beta8 and alpha2, alpha4, alpha6, alpha7 and alpha11 were never expressed. ILK, CD47 and ICAP1, as components of the intracellular signalling cascade of several integrins, were activated with ongoing dedifferentiation. In conclusion, a candidate for signal transmission during dedifferentiation is the fibronectin receptor (integrin alpha5beta1) in conjunction with its ligand fibronectin. Other receptors, e.g. for vitronectin and osteopontin (alphaVbeta3) or laminin (alpha6beta1) or their ligands, do not seem to be involved in signal transmission for dedifferentiation. In addition, the GPIIb/IIIa-receptor seems to assist the process of dedifferentiation. Intracellularly, ILK, ICAP1 and CD47 might assist the transduction of the integrin-dependent signals.
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Condrocitos/citología , Condrocitos/fisiología , Integrinas/genética , Ingeniería de Tejidos/métodos , Técnicas de Cultivo de Célula , División Celular , Matriz Extracelular/fisiología , Humanos , Tabique Nasal , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genéticaRESUMEN
CONCLUSIONS: With ongoing culture and dedifferentiation of chondrocytes, significant changes in the expression patterns of various collagens and the insulin-like growth factor (IGF) receptor were detected. The latter could play an important role in the differentiation of human chondrocytes. OBJECTIVE: Tissue engineering represents a promising method for the construction of autologous chondrogenic grafts for reconstructive surgery. So far, little is known about the expression of markers for cell proliferation and differentiation in cultured chondrocytes. MATERIAL AND METHODS: Human chondrocytes were isolated from septal cartilage (n=5) and held in primary cell culture. Cells were harvested after 24 h and 6 days. Proliferation was analyzed using an Alamar Blue assay. The differentiation of the cells was investigated using bright field microscopy, the expression patterns of various proteins using immunohistochemistry and the expression of distinct genes using a microarray technique. RESULTS: The chondrocytes showed strong proliferation (Day 0: 16.7+/-0.7 fluorescent units; Day 5: 52.4+/-2.2 fluorescent units) from the third day of cell culture in medium without growth factors. From this point onwards, a dedifferentiation of the chondrocytes could be observed. In cell culture, the chondrocytes expressed collagen 1 and 10 without expression of collagen 3. After 6 days of cell culture, they expressed collagen 2. The chondrocytes showed constant low expression of the fibroblast growth factor-2 receptor, but constant high expression of vascular endothelial growth factor, matrix metalloproteinase (MMP)2 and MMP9. The cells never expressed the epidermal growth factor receptor. The proportion of IGF receptor-expressing cells diminished significantly during cell culture.
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Cartílago/citología , Condrocitos/metabolismo , Proteínas de la Matriz Extracelular/análisis , Sustancias de Crecimiento/análisis , Ingeniería de Tejidos , Cartílago/fisiología , Diferenciación Celular , Proliferación Celular , Separación Celular , Células Cultivadas , Colágeno Tipo I/análisis , Colágeno Tipo II/análisis , Colágeno Tipo III/análisis , Colágeno Tipo X/análisis , Receptores ErbB/análisis , Factor 2 de Crecimiento de Fibroblastos/análisis , Humanos , Metaloproteinasa 2 de la Matriz/análisis , Metaloproteinasa 9 de la Matriz/análisis , Tabique Nasal/citología , Receptor IGF Tipo 1/análisis , Receptores de Factores de Crecimiento de Fibroblastos/análisis , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/análisisRESUMEN
Tissue engineering represents a promising method for the construction of autologous chondrogenic grafts for reconstructive surgery. The destruction or malformation of organs such as nasal cartilage, pinna and trachea in otorhinology-head and neck surgery can be caused by both: primary disease or treatment modalities. A large part of modern medical practice is aimed to repair, replace, maintain or enhance the function of damaged or diseased tissues and organs. Replacement or repair is by either artificial implants or transplantation of tissues. Such interventions are hindered by factors such as rejection by the immune system, limited blood supply or morbidity of the donor site. Reconstruction of an injured face using plastic surgery is a prime example of when the limitations of materials, science and reconstructive techniques become apparent. This review aims to briefly outline the use of chondrocytes for tissue engineering with special regard to the function of the extracellular matrix for the signalling between the chondrocytes.
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Condrocitos/citología , Matriz Extracelular/fisiología , Ingeniería de Tejidos/métodos , Animales , Anexina A5/farmacología , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular , División Celular , Condrocitos/metabolismo , Condrocitos/patología , Colorantes/farmacología , Matriz Extracelular/metabolismo , Sustancias de Crecimiento/metabolismo , Humanos , Modelos Biológicos , Regeneración , Transducción de Señal , Somatomedinas/metabolismo , Células Madre/patología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Chondrocytes surrounded by extracellular matrix are responsible for the maintenance of the cartilage as a functional entity. It is well accepted that chondrocytes cultivated for tissue engineering dedifferentiate in cell culture. We characterized the expression of different collagens and collagen related proteins in differentiated (primary) and cultured nasal chondrocytes by using microarray gene expression analysis and immunohistochemical staining. The genes for collagen subunits 1alpha1 (Col1alpha1) and 1alpha2 (Col1alpha2) were activated during a cell culture period of 5 and 20 days whereas Col2alpha1 could be detected both in differentiated and dedifferentiated chondrocytes. The long-term cell culture revealed a late activation of the Col3alpha1, Col4alpha1 and Col11alpha1 genes as well as biglycan, fibromodulin and lumican. In addition, short- and long-term cell culture resulted in down-regulation of Col9alpha1, Col9alpha2, Col9alpha3, Col10alpha1, Col18alpha1, ColQ and chondroadherin. The decorin gene showed up-regulation in short-term cell culture, but down-regulation in long-term culture. Immunohistochemical staining of the different cell populations confirmed the mRNA data for collagen type 1, 2, 3, 4, 9alpha2, 9alpha3, 18 and decorin. Because of their up-regulation in cultured chrondrocytes the collagen types 1, 3, 4 and 11 as well as biglycan, fibromodulin and lumican may be markers for dedifferentiation. The collagen types 9, 18 and Q as well as decorin and chondro-adherin revealed down-regulation and, presumably, represent markers for the differentiation of chondrocytes.
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Cartílago/citología , Cartílago/metabolismo , Diferenciación Celular , Colágeno/genética , Colágeno/metabolismo , Perfilación de la Expresión Génica , Biglicano , Células Cultivadas , Condrocitos/citología , Condrocitos/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/genética , Decorina , Proteínas de la Matriz Extracelular/genética , Fibromodulina , Humanos , Inmunohistoquímica , Sulfato de Queratano/genética , Lumican , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteoglicanos/genética , Proteoglicanos/metabolismo , ARN Mensajero/análisis , ARN Mensajero/genéticaRESUMEN
Tissue engineering is a promising research field, which aims to create new functional muscle tissue in vitro, by utilizing the myogenic differentiation potential of human stem cells. The objective of the present study was to determine the effect of static magnetic fields (SMF), combined with the use of the myogenic differentiation enhancing hepatocyte growth factor (HGF), on human satellite cell cultures, which are one of the preferred stem cell sources in skeletal muscle tissue engineering. We performed almarBlue® proliferation assays and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) for the following myogenic markers: desmin (DES), myogenic factor 5 (MYF5), myogenic differentiation antigen 1 (MYOD1), myogenin (MYOG), myosin heavy chain (MYH) and α1 actin (ACTA1) to detect the effects on myogenic maturation. Additionally, immunohistochemical staining (ICC) and fusion index (FI) determination as independent markers of differentiation were performed on satellite cell cultures stimulated with HGF and HGF + SMF with an intensity of 80 mT. ICC verified the muscle phenotype at all time points. SMF enhanced the proliferation of satellite cell cultures treated with HGF. RT-PCR analysis, ICC and FI calculation revealed the effects of HGF/SMF on the investigated differentiation markers and stimulation with HGF and SMF verified the continuing maturation, however no significant increase in analysed markers could be detected when compared with control cultures treated with serum cessation. In conclusion, HGF or HGF + SMF stimulation of human satellite cell cultures did not lead to the desired enhancement of myogenic maturation of human satellite cell cultures compared with cell cultures stimulated with growth factor reduction.
Asunto(s)
Factor de Crecimiento de Hepatocito/farmacología , Campos Magnéticos , Células Satélite del Músculo Esquelético/efectos de los fármacos , Células Satélite del Músculo Esquelético/efectos de la radiación , Actinas/genética , Actinas/metabolismo , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Células Cultivadas , Desmina/genética , Desmina/metabolismo , Expresión Génica , Humanos , Proteína MioD/genética , Proteína MioD/metabolismo , Factor 5 Regulador Miogénico/genética , Factor 5 Regulador Miogénico/metabolismo , Miogenina/genética , Miogenina/metabolismo , Células Satélite del Músculo Esquelético/metabolismoRESUMEN
The creation of functional muscles/muscle tissue from human stem cells is a major goal of skeletal muscle tissue engineering. Mesenchymal stem cells (MSCs) from fat/adipose tissue (AT-MSCs), as well as bone marrow (BM-MSCs) have been shown to bear myogenic potential, which makes them candidate stem cells for skeletal muscle tissue engineering applications. The aim of this study was to analyse the myogenic differentiation potential of human AT-MSCs and BM-MSCs cultured in six different cell culture media containing different mixtures of growth factors. The following cell culture media were used in our experiments: mesenchymal stem cell growth medium (MSCGM)™ as growth medium, MSCGM + 5-azacytidine (5-Aza), skeletal muscle myoblast cell growth medium (SkGM)-2 BulletKit™, and 5, 30 and 50% conditioned cell culture media, i.e., supernatant of human satellite cell cultures after three days in cell culture mixed with MSCGM. Following the incubation of human AT-MSCs or BM-MSCs for 0, 4, 8, 11, 16 or 21 days with each of the cell culture media, cell proliferation was measured using the alamarBlue® assay. Myogenic differentiation was evaluated by quantitative gene expression analyses, using quantitative RT-PCR (qRT-PCR) and immunocytochemical staining (ICC), using well-defined skeletal markers, such as desmin (DES), myogenic factor 5 (MYF5), myosin, heavy chain 8, skeletal muscle, perinatal (MYH8), myosin, heavy chain 1, skeletal muscle, adult (MYH1) and skeletal muscle actin-α1 (ACTA1). The highest proliferation rates were observed in the AT-MSCs and BM-MSCs cultured with SkGM-2 BulletKit medium. The average proliferation rate was higher in the AT-MSCs than in the BM-MSCs, taking all six culture media into account. qRT-PCR revealed the expression levels of the myogenic markers, ACTA1, MYH1 and MYH8, in the AT-MSC cell cultures, but not in the BM-MSC cultures. The muscle-specific intermediate filament, DES, was only detected (by ICC) in the AT-MSCs, but not in the BM-MSCs. The strongest DES expression was observed using the 30% conditioned cell culture medium. The detection of myogenic markers using different cell culture media as stimuli was only achieved in the AT-MSCs, but not in the BM-MSCs. The strongest myogenic differentiation, in terms of the markers examined, was induced by the 30% conditioned cell culture medium.
Asunto(s)
Tejido Adiposo/citología , Diferenciación Celular/efectos de los fármacos , Medios de Cultivo/química , Células Madre Mesenquimatosas/efectos de los fármacos , Desarrollo de Músculos/efectos de los fármacos , Músculo Esquelético/citología , Actinas/genética , Actinas/metabolismo , Tejido Adiposo/efectos de los fármacos , Azacitidina/farmacología , Células de la Médula Ósea/citología , Técnicas de Cultivo de Célula , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo/farmacología , Desmina/genética , Desmina/metabolismo , Humanos , Músculo Esquelético/efectos de los fármacos , Factor 5 Regulador Miogénico/genética , Factor 5 Regulador Miogénico/metabolismo , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismoRESUMEN
The cancer stem cell (CSC) theory implies that CSCs are surrounded by supportive stromal cells, which are known as the CSC niche. Stromal cell-derived factor-1 (SDF-1) shows a multitude of functional effects in head and neck squamous cell carcinoma (HNSCC) cells, including migration and polarization. Therefore, the SDF-1-CXCR4 axis may be involved in the pathophysiology of the progression, recurrence and metastasis of malignant diseases of the head and neck. In the present study, the CD44+ HNSCC UM-SCC-11A cell line was used as a model for CSCs. The interaction between the UM-SCC-11A cells and the supportive microenvironmental cells, including fibrocytes, human umbilical vein endothelial cells (HUVECs) and human microvascular vein endothelial cells (HMVECs) was evaluated. All the cell types that were tested were shown to secrete different concentrations of SDF-1 into the surrounding culture medium [mean (m)fibro, 1243.3±156.2 pg/ml; mHMVEC, 1061.4±23.2 pg/ml; mHUVEC, 849.6±110.9 pg/ml]. The migration of the UM-SCC-11A cells towards the supportive cells was increased by a higher supply of SDF-1 (contrfibro, 315.23±61.55 µm; mfibro, 477.73±143.7 µm; Pfibro=0.003; contrHMVEC, 123.41±66.68 µm; mHMVEC, 249.04±111.95 µm; PHMVEC=0.004; contrHUVEC, 189.7±93.26 µm; mHUVEC, 260.82±161.58 µm). The amount of the UM-SCC-11A cells that migrated towards the differentiated fibrocytes was significantly higher than that which migrated towards the HMVECs or HUVECs (Pfibro/HMVEC=2.12E-11; Pfibro/HUVEC=2.28E-5). Cell-cell interaction by podia formation of the UM-SCC-11A cells was observed in all the supportive cell types that were tested. Broadly based cell-cell contacts were observed. By contrast, digitiform podia formations presented by the UM-SCC-11A cells were determined using fluorescence microscopy. The SDF-1-CXCR4 axis is postulated to be a crucial pathway in the interaction between CSCs and their surrounding supportive cells. Understanding the cell-cell interactions in the CSC niche using in vitro models may aid in gaining further insight into these mechanisms and finding new strategies of therapy in this field.
RESUMEN
Stromal cell-derived factor-1α (SDF-1α), also known as CXCL12, has variable effects on a plurality of cells. It is known to have selective effects on cell migration, morphology, survival and cell homing. As such the SDF-1-CXCR4 axis is postulated to be a crucial key pathway in the interaction between (cancer) stem cells and their surrounding supportive cells, the so-called (cancer) stem cell niche. We evaluated the expression of CD44 as a cancer stem cell (CSC) marker and the expression of CXCR4 in the head and neck squamous cell carcinoma (HNSCC) cell line UM-SCC 11A. In addition, we monitored proliferation, formation of podia and migration of UM-SCC 11A cells under the influence of SDF-1α. Whereas SDF-1α induced the formation of podia of CD44(+) CXCR4(+) UM-SCC 11A cells in a dose-dependent manner and the maximum number of cells exhibiting the formation of podia was observed under the influence of 10 ng/ml SDF-1α (P=5.3x10(-6)), the highest number of migrating cells was noted using a concentration of 100 ng/ml (P=0.027). Proliferation and survival were not affected by SDF-1α. We showed that UM-SCC 11A cells could be a target for SDF-1α by CXCR4 expression and these cells also showed characteristics of HNSCC CSCs via CD44 expression. We demonstrated that SDF-1α is a chemoattractant for UM-SCC 11A cells, and a maximum directed migration was achieved under the influence of 100 ng/ml SDF-1α. Changes in cell morphology by presenting filopodia or a prominent uropod were noted following treatment of 10 ng/ml SDF-1α. The SDF-CXCR4 axis may play a crucial role in the interaction between CSCs and their supportive cells in the CSC niche. Understanding these interactions may help to gain further insight into the pathophysiology of the progression and recurrence of malignant diseases and thus help to develop novel strategies for therapy.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Quimiocina CXCL12/farmacología , Neoplasias de Cabeza y Cuello/metabolismo , Receptores de Hialuranos/metabolismo , Receptores CXCR4/metabolismo , Línea Celular Tumoral , Polaridad Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Humanos , Modelos Biológicos , Células Madre Neoplásicas , Seudópodos/efectos de los fármacos , Transducción de Señal , Nicho de Células MadreRESUMEN
Stromal cell-derived factor-1α (SDF-1α), also known as CXCL12, has variable effects on a plurality of cells. CXCR4 has been identified as its corresponding receptor. The SDF-1-CXCR4 axis is postulated to be a crucial key pathway in the interaction between (cancer) stem cells and their surrounding supportive cells in the cancer stem cell niche. We evaluated the expression of CD44 as a cancer stem cell marker and of CXCR4 in human HNSCC tissue samples. Afterwards, we monitored the concentration of SDF-1 in peripheral blood samples of HNSCC patients and healthy donors. We showed that CD44 and CXCR4 are expressed in human HNSCC tissues. Markedly, CD44 showed a high expression in HNSCC cells bordering cancer stromal cells. CXCR4 was mainly expressed in HNSCC tumor nests, but not in the surrounding stromal cells. No significant difference was noted between the SDF-1 concentration in the peripheral blood of HNSCC patients compared to healthy donors. We showed that CD44, as a stem cell marker in HNSCC, is located mainly at the borderline of HNSCC tumor nests with the surrounding cells. In addition, we demonstrated that CXCR4 as the corresponding receptor to SDF-1 is highly expressed in HNSCC tumor nests, but not in the tumor stroma. We collected evidence that SDF-1-CXCR4 interaction may be a crucial pathway in cell trafficking in the cancer stem cell niche of HNSCC, while SDF-1 was not detected in the peripheral blood of HNSCC patients. The SDF-1-CXCR4 axis may play an important role in the cancer stem cell theory of HNSCC. As SDF-1α also exhibits a multitude of functional effects on HNSCC cells, such as migration and polarization, it may be possible that the SDF-1-CXCR4 axis is also involved in the pathophysiology of the progression, recurrence and metastasis of malignant disease. Understanding these interactions may help to gain further insight into these mechanisms and as such help to discover new strategies of therapy.
Asunto(s)
Carcinoma de Células Escamosas/sangre , Quimiocina CXCL12/sangre , Células Madre Neoplásicas/metabolismo , Neoplasias de Oído, Nariz y Garganta/sangre , Receptores CXCR4/metabolismo , Nicho de Células Madre , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/patología , Estudios de Casos y Controles , Movimiento Celular , Femenino , Humanos , Receptores de Hialuranos/metabolismo , Masculino , Persona de Mediana Edad , Neoplasias de Oído, Nariz y Garganta/patologíaRESUMEN
Tissue engineering of skeletal muscle is an encouraging possibility for the treatment of muscle loss through the creation of functional muscle tissue in vitro from human stem cells. Currently, the preferred stem cells are primary, non-immunogenic satellite cells ( = myoblasts). The objective of this study was to determine the expression patterns of myogenic markers within the human satellite cell population during their differentiation into multinucleated myotubes for an accurate characterization of stem cell behaviour. Satellite cells were incubated (for 1, 4, 8, 12 or 16 days) with a culture medium containing either a low [ = differentiation medium (DM)] or high [ = growth medium (GM)] concentration of growth factors. Furthermore, we performed a quantitative gene expression analysis of well-defined differentiation makers: myogenic factor 5 (MYF5), myogenin (MYOG), skeletal muscle αactin1 (ACTA1), embryonic (MYH3), perinatal (MYH8) and adult skeletal muscle myosin heavy chain (MYH1). Additionally, the fusion indices of forming myotubes of MYH1, MYH8 and ACTA1 were calculated. We show that satellite cells incubated with DM expressed multiple characteriztic features of mature skeletal muscles, verified by time-dependent upregulation of MYOG, MYH1, MYH3, MYH8 and ACTA1. However, satellite cells incubated with GM did not reveal all morphological aspects of muscle differentiation. Immunocytochemical investigations with antibodies directed against the differentiation markers showed correlations between the gene expression and differentiation. Our data provide information about time-dependent gene expression of differentiation markers in human satellite cells, which can be used for maturation analyses in skeletal muscle tissue-engineering applications.