RESUMEN
The current gold standard in peripheral nerve repair is nerve autografts for bridging gaps larger than a centimeter. However, autografts are associated with a low availability and the loss of function at the donor site. Nerve guidance conduits (NGCs) made of biocompatible and biodegradable materials reflect suitable alternatives. Clinically approved NGCs comprise either wraps that are rolled around the loose ends of the nerve or steady-state tubes; however, both lack internal guidance structures. Here, we established self-rolling NGCs to allow for gentle encapsulation of nerve cells together with supportive microenvironments, such as (1) an inner tube wall coating with a bioactive spider silk film, (2) an inner tube wall lining using an anisotropic spider silk non-woven mat, or (3) a luminal filler using an anisotropic collagen cryogel. Neuronal cells adhered and differentiated inside the modified tubes and formed neurites, which were oriented along the guidance structures provided by the spider silk non-woven mat or by the fibrillary structure of the collagen cryogel. Thus, our size-adaptable NGCs provide several features useful for peripheral nerve repair, and distinct combinations of the used elements might support and enhance the clinical outcome.
RESUMEN
Objective. To investigate the expression and target genes of pigment epithelium-derived factor (PEDF) in cartilage and chondrocytes, respectively. Methods. We analyzed the expression pattern of PEDF in different human cartilaginous tissues including articular cartilage, osteophytic cartilage, and fetal epiphyseal and growth plate cartilage, by immunohistochemistry and quantitative real-time (qRT) PCR. Transcriptome analysis after stimulation of human articular chondrocytes with rhPEDF was performed by RNA sequencing (RNA-Seq) and confirmed by qRT-PCR. Results. Immunohistochemically, PEDF could be detected in transient cartilaginous tissue that is prone to undergo endochondral ossification, including epiphyseal cartilage, growth plate cartilage, and osteophytic cartilage. In contrast, PEDF was hardly detected in healthy articular cartilage and in the superficial zone of epiphyses, regions that are characterized by a permanent stable chondrocyte phenotype. RNA-Seq analysis and qRT-PCR demonstrated that rhPEDF significantly induced the expression of a number of matrix-degrading factors including SAA1, MMP1, MMP3, and MMP13. Simultaneously, a number of cartilage-specific genes including COL2A1, COL9A2, COMP, and LECT were among the most significantly downregulated genes. Conclusions. PEDF represents a marker for transient cartilage during all neonatal and postnatal developmental stages and promotes the termination of cartilage tissue by upregulation of matrix-degrading factors and downregulation of cartilage-specific genes. These data provide the basis for novel strategies to stabilize the phenotype of articular cartilage and prevent its degradation.
Asunto(s)
Cartílago/metabolismo , Cartílago/patología , Condrocitos/metabolismo , Condrocitos/patología , Proteínas del Ojo/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Serpinas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Epífisis/metabolismo , Proteínas del Ojo/genética , Feto/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Placa de Crecimiento/metabolismo , Humanos , Inmunohistoquímica , Articulaciones/metabolismo , Articulaciones/patología , Factores de Crecimiento Nervioso/genética , Osteofito/genética , Osteofito/patología , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Serpinas/genética , Transducción de Señal/genéticaRESUMEN
Objective and sensitive assessment of cartilage repair outcomes lacks suitable methods. This study investigated the feasibility of 3D ultrasound biomicroscopy (UBM) to quantify cartilage repair outcomes volumetrically and their correlation with established classification systems. 32 sheep underwent bilateral treatment of a focal cartilage defect. One or two years post-operatively the repair outcomes were assessed and scored macroscopically (Outerbridge, ICRS-CRA), by magnetic resonance imaging (MRI, MOCART), and histopathology (O'Driscoll, ICRS-I and ICRS-II). The UBM data were acquired after MRI and used to reconstruct the shape of the initial cartilage layer, enabling the estimation of the initial cartilage thickness and defect volume as well as volumetric parameters for defect filling, repair tissue, bone loss and bone overgrowth. The quantification of the repair outcomes revealed high variations in the initial thickness of the cartilage layer, indicating the need for cartilage thickness estimation before creating a defect. Furthermore, highly significant correlations were found for the defect filling estimated from UBM to the established classification systems. 3D visualisation of the repair regions showed highly variable morphology within single samples. This raises the question as to whether macroscopic, MRI and histopathological scoring provide sufficient reliability. The biases of the individual methods will be discussed within this context. UBM was shown to be a feasible tool to evaluate cartilage repair outcomes, whereby the most important objective parameter is the defect filling. Translation of UBM into arthroscopic or transcutaneous ultrasound examinations would allow non-destructive and objective follow-up of individual patients and better comparison between the results of clinical trials.
Asunto(s)
Huesos/diagnóstico por imagen , Cartílago Articular , Microscopía Acústica/métodos , Animales , Desarrollo Óseo/fisiología , Huesos/citología , Cartílago Articular/diagnóstico por imagen , Cartílago Articular/lesiones , Cartílago Articular/cirugía , Método Doble Ciego , Femenino , Estudios Prospectivos , Distribución Aleatoria , Reproducibilidad de los Resultados , Ovinos , Cicatrización de Heridas/fisiologíaRESUMEN
OBJECTIVE: The initiation/progression factors of osteoarthritic (OA) cartilage degeneration and the involved biological mechanisms remain rather enigmatic. One core reason for this might be a cellular senescence-like phenotype of OA chondrocytes, which might show a fundamentally different behavior pattern unexpected from the biological mechanism established in young cells. DESIGN: This study was designed to investigate one core property of senescent cells, the heterogeneity of gene expression, in OA chondrocytes by double-labeling immunolocalization using two genes (vimentin, S-100 protein) as surrogates, which are constitutively expressed by (normal) chondrocytes. The level of genomic DNA damage in OA chondrocytes was compared to normal chondrocytes and in vitro experiments designed to demonstrate that stochastic genomic DNA damage is able to induce heterogeneity of gene expression in chondrocytes. RESULTS: We show a significantly increased heterogeneity of gene expression for vimentin and S-100 protein as well as a significantly increased genomic DNA damage in the OA compared to normal chondrocytes, whereas no evidence of critical telomere shortening was found. In vitro experiments demonstrated that stochastic genomic DNA damage induced by increased oxidative or genotoxic stress is able to induce the heterogeneity in gene expression found in the OA cells in situ. CONCLUSIONS: Our results suggest that OA chondrocytes show a special form of age-related cell degeneration, "progressive/stress-induced senescence", progressing over time due to accumulated DNA damage and subsequent chaotic gene activation pattern. This promotes increased malfunctioning of the cells and finally the loss of their capacity to keep up cell and tissue homeostasis, i.e., prevent OA.
Asunto(s)
Cartílago Articular/metabolismo , Senescencia Celular/genética , Condrocitos/metabolismo , Expresión Génica , Osteoartritis de la Rodilla/genética , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Daño del ADN , Citometría de Flujo , Humanos , Persona de Mediana Edad , Osteoartritis de la Rodilla/metabolismo , Proteínas S100/metabolismo , Vimentina/metabolismoRESUMEN
Degenerative disorders of the musculoskeletal system, in particular osteoarthritis, are among the most common diseases of the elderly and their importance in an aging society is continuously increasing. Correspondingly, many surgical interventions are undertaken and pathological specimens submitted for histopathologic workup. The pathophysiology of osteoarthritis, which ultimately leads to joint destruction, is still poorly understood. The question remains as to whether the cause lies (mainly) within the chondrocytes themselves (e.g. cellular aging/senescence) or whether the synovial membrane or the subchondral bone are equally or even more important factors. The process of joint destruction can be evaluated in terms of pathogenesis (typing), extent (staging) and degree of the most extensive focal damage (grading). Because of the heterogeneity of the disease and substantial individual differences in progression, classification and grading of cartilage degeneration represents a complex task. Any pathology report should be concise and delineate only the essential features. Differentiating between primary osteoarthritis and secondary degenerative changes, e.g., due to previously unknown rheumatoid disease, bone necrosis or an infection of the joint, is of particular clinical interest.
Asunto(s)
Osteoartritis/etiología , Osteoartritis/patología , Anciano , Huesos/patología , Cartílago Articular/patología , Senescencia Celular/fisiología , Condrocitos/patología , Humanos , Osteoartritis/clasificación , Membrana Sinovial/patologíaRESUMEN
Cell therapies for articular cartilage defects rely on expanded chondrocytes. Mesenchymal stem cells (MSC) represent an alternative cell source should their hypertrophic differentiation pathway be prevented. Possible cellular instruction between human articular chondrocytes (HAC) and human bone marrow MSC was investigated in micromass pellets. HAC and MSC were mixed in different percentages or incubated individually in pellets for 3 or 6 weeks with and without TGF-beta1 and dexamethasone (±T±D) as chondrogenic factors. Collagen II, collagen X and S100 protein expression were assessed using immunohistochemistry. Proteoglycan synthesis was evaluated applying the Bern score and quantified using dimethylmethylene blue dye binding assay. Alkaline phosphatase activity (ALP) was detected on cryosections and soluble ALP measured in pellet supernatants. HAC alone generated hyaline-like discs, while MSC formed spheroid pellets in ±T±D. Co-cultured pellets changed from disc to spheroid shape with decreasing number of HAC, and displayed random cell distribution. In -T-D, HAC expressed S100, produced GAG and collagen II, and formed lacunae, while MSC did not produce any cartilage-specific proteins. Based on GAG, collagen type II and S100 expression chondrogenic differentiation occurred in -T-D MSC co-cultures. However, quantitative experimental GAG and DNA values did not differ from predicted values, suggesting only HAC contribution to GAG production. MSC produced cartilage-specific matrix only in +T+D but underwent hypertrophy in all pellet cultures. In summary, influence of HAC on MSC was restricted to early signs of neochondrogenesis. However, MSC did not contribute to the proteoglycan deposition, and HAC could not prevent hypertrophy of MSC induced by chondrogenic stimuli.
Asunto(s)
Células de la Médula Ósea/metabolismo , Cartílago Articular/citología , Diferenciación Celular , Condrocitos/citología , Células Madre Mesenquimatosas/citología , Células de la Médula Ósea/citología , Cartílago Articular/metabolismo , Células Cultivadas , Condrocitos/metabolismo , Técnicas de Cocultivo , Colágeno Tipo II/metabolismo , Humanos , Células Madre Mesenquimatosas/metabolismo , Fenotipo , Proteoglicanos/biosíntesis , Proteínas S100/metabolismoRESUMEN
Mesenchymal stem cells (MSC) are increasingly replacing chondrocytes in tissue engineering based research for treatment of osteochondral defects. The aim of this work was to determine whether repair of critical-size chronic osteochondral defects in an ovine model using MSC-seeded triphasic constructs would show results comparable to osteochondral autografting (OATS). Triphasic implants were engineered using a beta-tricalcium phosphate osseous phase, an intermediate activated plasma phase, and a collagen I hydrogel chondral phase. Autologous MSCs were used to seed the implants, with chondrogenic predifferentiation of the cells used in the cartilage phase. Osteochondral defects of 4.0 mm diameter were created bilaterally in ovine knees (n = 10). Six weeks later, half of the lesions were treated with OATS and half with triphasic constructs. The knees were dissected at 6 or 12 months. With the chosen study design we were not able to demonstrate significant differences between the histological scores of both groups. Subcategory analysis of O'Driscoll scores showed superior cartilage bonding in the 6-month triphasic group compared to the autograft group. The 12-month autograft group showed superior cartilage matrix morphology compared to the 12-month triphasic group. Macroscopic and biomechanical analysis showed no significant differences at 12 months. Autologous MSC-seeded triphasic implants showed comparable repair quality to osteochondral autografts in terms of histology and biomechanical testing.
Asunto(s)
Cartílago Articular/lesiones , Trasplante de Células Madre Mesenquimatosas/métodos , Ingeniería de Tejidos/métodos , Andamios del Tejido , Animales , Cartílago/trasplante , Condrocitos/trasplante , Femenino , OvinosRESUMEN
Histological and histochemical methods are important tools in the evaluation of joint tissue samples for degenerative joint diseases, both in humans and in animal models. In this respect, standardized, simple, and reliable techniques are mandatory. This chapter describes five basic staining procedures appropriate for macroscopic (Indian ink) and histologic (HE/hematoxylin - eosin) visualization and scoring of cartilage proteoglycan and collagen content (toluidine blue/safranin O and picrosirius red/Goldner's trichrome).
Asunto(s)
Artritis Experimental/patología , Osteoartritis/patología , Animales , Artritis Experimental/metabolismo , Colágeno/metabolismo , Modelos Animales de Enfermedad , Técnicas de Preparación Histocitológica/métodos , Humanos , Osteoartritis/metabolismo , Proteoglicanos/metabolismo , Coloración y Etiquetado/métodosRESUMEN
Animal model systems represent an important adjunct and surrogate for studies of osteoarthritis (OA) in humans. They provide a means to study OA pathophysiology as well as aid in the development of therapeutic agents and biological markers for diagnosing and prognosing the disease. Thus, it is of great importance for the OA scientific community, both in academic as well as industrial research, to standardize scoring systems for evaluating the OA disease process and to make results between different studies comparable. The task of the histopathology initiative of OARSI was to achieve a consensus of scoring systems for the most important species used in OA animal model research (dog, guinea pig, horse, mouse, rabbit, rat, and sheep/goat), which are presented in the various chapters in this special volume of Osteoarthritis & Cartilage together with extra chapters on basic methodology (histochemistry, statistics, morphometry), the specific terminology and a general discussion of animal models in OA research. Standardized definitions are suggested for basic but essential terms such as "grading" and "staging" in order to promote their consistent use and thereby promote improved understanding and data interpretation across all model systems. Thus, this introductory chapter presents an overview of the guiding principles for assessment of important OA animal model systems. Use of such systems, independently or in conjunction with other systems in parallel, should facilitate comparability of results across animal model studies.
Asunto(s)
Artritis Experimental/patología , Atlas como Asunto , Modelos Animales de Enfermedad , Osteoartritis/patología , Índice de Severidad de la Enfermedad , Animales , Conferencias de Consenso como Asunto , Especificidad de la Especie , Terminología como AsuntoRESUMEN
Unifying terminology used to describe morphologic features is a very important endeavour to assure comparability of work and papers on osteoarthritis animal models. In this editorial an attempt is presented to define and unify the terminology of the macroscopic and histological description of joint changes in OA for both human OA and the OA animal models.
Asunto(s)
Artritis Experimental/patología , Cartílago Articular/patología , Articulaciones/patología , Osteoartritis/patología , Terminología como Asunto , Animales , Condrocitos/patología , Modelos Animales de Enfermedad , HumanosRESUMEN
Nora's lesion, also known as "bizarre parosteal osteochondromatous proliferation" (BPOP), was first described in 1983 by the pathologist Nora. This lesion is defined as a proliferation of the bone. In most cases the lesion emanates from the intact cortical substance of short bones. It used to be assigned to reactive, heterotopic ossifications. More recent publications described constant genetic alterations supposing a tumorous genesis. Nora lesions are mostly found in the third or fourth decade of life; a preference of sexes is not described in the literature. They are characterized by a typical appearance in radiological diagnostics, but the diagnosis is ultimately determined by histopathological examination. Surgical resection is the therapy of choice.We report the case of a 29-year-old patient with an undetermined proliferation of the proximal ulna. The diagnosis of a Nora's lesion was made. The therapeutic approach, differential diagnosis and corresponding literature are presented and discussed.
Asunto(s)
Neoplasias Óseas/diagnóstico por imagen , Neoplasias Óseas/cirugía , Osteocondroma/diagnóstico por imagen , Osteocondroma/cirugía , Cúbito/diagnóstico por imagen , Cúbito/cirugía , Humanos , Masculino , Persona de Mediana Edad , Radiografía , Enfermedades Raras/diagnóstico por imagen , Enfermedades Raras/cirugíaRESUMEN
Osteoarthritis, the degeneration of the joints, is the leading source of physical disability with severely impaired quality of life due to pain and loss of joint functioning in industrialized nations. Clinically, degeneration affects mostly the large weight bearing joints of the legs like the hip or the knees, but in principle it can affect any joint of the body. Osteoarthritis represents a disease group with disease subsets that have different underlying pathophysiological mechanisms. Therefore primary osteoarthritis has to be distinguished from secondary forms of the disease, which are due to traumatic events, endocrine or metabolic disorders etc. The enormous frequency of this disease makes osteoarthritis one of the most expensive conditions in the Western world, both in terms of direct as well as indirect costs. So far, despite intensive efforts over several decades, the success of disease-modifying approaches have been rather limited and mostly restricted to analgesis and non-pharmacologic therapy (e.g. nonsteroidal anti-inflammatory agents, exercise, and physiotherapy). Joint replacement is still the unsurpassed therapy for the symptomatic relief of advanced and incapacitating OA. It is evident that there is a great need for the development of disease modifying agents in order to improve quality of life as well as to relieve the community of the enormous socio-economic burden of the disease.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Articulaciones/fisiología , Osteoartritis/fisiopatología , Matriz Ósea/metabolismo , Cartílago/metabolismo , Cartílago/patología , Condrocitos/metabolismo , Colágeno/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Cápsula Articular/patología , Articulaciones/anatomía & histología , Articulaciones/metabolismo , Modelos Biológicos , Osteoartritis/metabolismo , Osteoartritis/patología , Membrana Sinovial/metabolismo , Membrana Sinovial/patologíaRESUMEN
Cushing's syndrome can be caused by adrenocorticotropic hormone-secreting solid tumors. We report a rare case of an ileal endocrine carcinoma that produced ACTH and induced hypercortisolism. A now 47-year-old man presented at age 41 with weight gain, tremor, perspiration, and general fatigue. Laboratory testing showed hypercortisolism and diabetes mellitus. Further examinations revealed ectopic Cushing's syndrome. The search for the primary tumor was difficult. The patient underwent subtotal thyroidectomy and surgical removal of a pituitary lesion. After resection of an ACTH-producing metastasis of the mesentery, temporary remission of Cushing's syndrome ensued. At the age 45 the primary tumor was detected in the ileum by Ga-68 DOTATOC-PET scan and explorative laparotomy. After surgical removal of this well differentiated neuroendocrine carcinoma the patient significantly improved clinically. He experienced better blood pressure and remission of his diabetes mellitus in addition to increased muscular strength. Endocrine laboratory testing at follow-up examinations confirmed remission of hypercortisolism and diabetes mellitus. A Ga-68 DOTATOC PET scan and a 1 mg dexamethasone suppression test 5 months after surgery showed normal results. Ectopic ACTH secretion within the small bowel is very rare. This case underscores the difficulty in locating the source of ectopic ACTH secretion and suggests using small bowel barium study, tubus endoscopy or video endoscopy for preoperative localization if the small bowel is suspected as tumor source.
Asunto(s)
Síndrome de ACTH Ectópico/diagnóstico , Hormona Adrenocorticotrópica/metabolismo , Carcinoma/diagnóstico , Síndrome de Cushing/diagnóstico , Neoplasias del Íleon/diagnóstico , Tumores Neuroendocrinos/diagnóstico , Síndrome de ACTH Ectópico/etiología , Presión Sanguínea , Carcinoma/complicaciones , Carcinoma/metabolismo , Carcinoma/patología , Síndrome de Cushing/etiología , Diabetes Mellitus/etiología , Humanos , Neoplasias del Íleon/complicaciones , Neoplasias del Íleon/metabolismo , Neoplasias del Íleon/patología , Masculino , Mesenterio/cirugía , Persona de Mediana Edad , Fuerza Muscular , Tumores Neuroendocrinos/complicaciones , Tumores Neuroendocrinos/metabolismo , Tumores Neuroendocrinos/patología , Hipófisis/cirugíaRESUMEN
Perilesional changes of chronic focal osteochondral defects were assessed in the knees of 23 sheep. An osteochondral defect was created in the main load-bearing region of the medial condyle of the knees in a controlled, standardised manner. The perilesional cartilage was evaluated macroscopically and biopsies were taken at the time of production of the defect (T0), during a second operation one month later (T1), and after killing animals at three (T3; n = 8), four (T4; n = 8), and seven (T7; n = 8) months. All the samples were histologically assessed by the International Cartilage Repair Society grading system and Mankin histological scores. Biopsies were taken from human patients (n = 10) with chronic articular cartilage lesions and compared with the ovine specimens. The ovine perilesional cartilage presented with macroscopic and histological signs of degeneration. At T1 the International Cartilage Repair Society 'Subchondral Bone' score decreased from a mean of 3.0 (SD 0) to a mean of 1.9 (SD 0.3) and the 'Matrix' score from a mean of 3.0 (SD 0) to a mean of 2.5 (SD 0.5). This progressed further at T3, with the International Cartilage Repair Society 'Surface' grading, the 'Matrix' grading, 'Cell Distribution' and 'Cell Viability' grading further decreasing and the Mankin score rising from a mean of 1.3 (SD 1.4) to a mean of 5.1 (SD 1.6). Human biopsies achieved Mankin grading of a mean of 4.2 (SD 1.6) and were comparable with the ovine histology at T1 and T3. The perilesional cartilage in the animal model became chronic at one month and its histological appearance may be considered comparable with that seen in human osteochondral defects after trauma.
Asunto(s)
Cartílago Articular/patología , Osteocondritis/patología , Rótula/patología , Adolescente , Adulto , Animales , Artroscopía , Cartílago Articular/cirugía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Osteocondritis/cirugía , Rótula/cirugía , Ovinos , Adulto JovenRESUMEN
Osteoarthritis is one of the most common diseases in modern western societies, particularly in the elderly, but it is occurring more and more often in the younger and middle-aged population, especially after traumatic injuries. The classification and grading of changes during cartilage degeneration is difficult due to the notoriously high heterogeneity of the disease process and is only partly clinically relevant. Overall, the process of joint destruction can always be evaluated for the pathogenesis (typing), its extent (staging), and the degree of the most extensive focal damage (grading). However, in the clinical routine, description and reporting of the basic findings might be best restricted to specimens obtained from endoprosthetic surgery. Only the identification of previously unknown underlying conditions such as rheumatoid disease, gout, or extensive osteonecrosis is of particular clinical interest.
Asunto(s)
Biopsia/métodos , Osteoartritis/clasificación , Osteoartritis/patología , HumanosRESUMEN
The rare case of an osteoid osteoma in the distal phalanx of the 2nd toe resulting in painful enlargement and hypertrophy of the entire toe in a 12-year-old girl is discussed. The tumour was excised and the oversize of the toe was corrected by exarticulation of the distal phalanx. 18 months postoperatively the patient demonstrates normal function of her forefoot without complaints or signs of inflammation.
Asunto(s)
Neoplasias Óseas/cirugía , Osteoma Osteoide/cirugía , Falanges de los Dedos del Pie/cirugía , Neoplasias Óseas/diagnóstico , Neoplasias Óseas/patología , Niño , Femenino , Humanos , Hipertrofia , Imagen por Resonancia Magnética , Osteoma Osteoide/diagnóstico , Osteoma Osteoide/patología , Falanges de los Dedos del Pie/patologíaRESUMEN
Bone metastases are found in 29% of patients with metastatic malignant choroidal melanoma, which is associated with poor prognosis. However there are several reports about prolonged survival. The unusual case of a patient is described, who suffered from a melanoma with orbital invasion and survived more than 18 years. Metastases were found 12 years after initial therapy. Three palliative operations made a survival of further 7 years with high quality of life possible. Therefore moderately palliative operations are recommended in case of metastatic malignant choroidal melanoma.
Asunto(s)
Neoplasias de la Coroides/secundario , Neoplasias de la Coroides/cirugía , Melanoma/secundario , Melanoma/cirugía , Cuidados Paliativos/métodos , Neoplasias Cutáneas/cirugía , Neoplasias de la Coroides/diagnóstico , Humanos , Masculino , Melanoma/diagnóstico , Persona de Mediana Edad , Neoplasias Cutáneas/diagnósticoRESUMEN
OBJECTIVE: The development of a reliable high-throughput transfection protocol for primary human articular chondrocytes. METHODS: Primary human chondrocytes were isolated from adult knee cartilage by an optimized enzymatic digestion protocol and cultivated in high-density monolayer culture for 3-5 days. Isolated chondrocytes were transfected with a green fluorescent protein (GFP)-expressing reporter construct using amaxa's Nucleofector 96-well Shuttle System. Transfection efficiencies were measured by fluorescence activated cell sorting and cell viability was determined by an adenosine-5'-triphosphate (ATP) assay. siRNA oligonucleotides (against glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) were transfected into the cells using the optimized nucleofection protocol and mRNA knockdown values were determined by a branched-DNA assay. RESULTS: Transfection efficiencies of more than 70% of surviving cells were achieved routinely with the nucleofection protocol presented in this article. Cell viability 24h post transfection was around 80%. The cell number used per transfection was reduced to 2x10(5) per sample. In addition, the protocol proved to be well suited for the transfer of siRNA molecules into primary human chondrocytes with suppression rates on the mRNA level of more than 95% (for GAPDH). CONCLUSIONS: We present the successful use of nucleofection on primary human chondrocytes using a microtiter plate compatible format that for the first time allows the efficient transfection of up to 96 samples in parallel. The optimized nucleofection protocol is offering maximum substrate flexibility by allowing transfer of DNA and siRNA oligonucleotides with the same set of parameters. Moreover, the transfection procedure requires substantially lower cell numbers than single cuvette protocols and is therefore perfectly suited for applications requiring multiple experimental replicates.
Asunto(s)
Cartílago Articular/patología , Condrocitos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Articulación de la Rodilla/patología , Transfección , Células Cultivadas , Citometría de Flujo/métodos , Proteínas Fluorescentes Verdes/genética , HumanosRESUMEN
OBJECTIVE: In this study, we were interested in the overall methylation level in aged and degenerated cartilage. Also, we looked at one gene which might be involved in the re-initiation of replicative activity in osteoarthritis (OA) chondrocytes, p21(WAF1/CIP1). p21(WAF1/CIP1) was previously suggested to be down-regulated in OA chondrocytes and is known to be regulated by epigenetic modulation. METHODS: Total methylation levels were analyzed by high pressure liquid chromatography (HPLC), mRNA expression of p21(WAF1/CIP1) and DNMT enzymes by real-time polymerase chain reaction. The methylation status of the p21(WAF1/CIP1)- promotor using bisulfite genomic sequencing was evaluated. RESULTS: General methylation analysis of genomic DNA showed no difference in between normal and aged/OA chondrocytes. Also no difference in methylation of the promotor of the p21(WAF1/CIP1) gene was detectable, which was significantly down-regulated in OA chondrocytes. DNMT1 and DNMT3a were expressed with no significant changes of expression levels found in OA chondrocytes. CONCLUSION: Cell cycle progression inhibitor p21(WAF1/CIP1) is expressed in normal and significantly down-regulated in OA articular chondrocytes, which may mediate the re-initiation of cell proliferation in OA cartilage. However, the suppression of p21(WAF1/CIP1) mRNA expression is not due to hypermethylation of its promotor. No overall changes in genome methylation levels were found in aged or OA cartilage. Interestingly, significant expression of DNA methyltransferases was found in articular chondrocytes, which supports that DNA methylation could still be a relevant mechanism of gene regulation in (osteoarthritic) chondrocytes, though not on an overall genomic level nor specifically for the regulation of the p21(WAF1/CIP1) gene.
Asunto(s)
Cartílago Articular/metabolismo , Condrocitos/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Metilación de ADN , Osteoartritis de la Rodilla/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Cartílago Articular/patología , Cromatografía Líquida de Alta Presión/métodos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , Regulación hacia Abajo/genética , Genoma , Humanos , Persona de Mediana Edad , Osteoartritis de la Rodilla/genética , Osteoartritis de la Rodilla/patología , Regiones Promotoras Genéticas , ARN Mensajero/genéticaRESUMEN
AIMS: Eosinophil infiltration of the oesophageal epithelium is the cardinal pathomorphological finding in eosinophilic oesophagitis (EO), but gastro-oesophageal reflux disease (GORD) is also associated with increased eosinophils. The aim was to compare histological parameters for the diagnosis of EO versus GORD on routinely taken biopsy specimens. METHODS AND RESULTS: One hundred and five routine biopsy specimens with EO (n = 62), GORD (n = 24) and probable EO (n = 19) from 74 patients (52 men, 22 women; mean age 43.7 years) were analysed for numbers of eosinophils, mast cells, degranulation and qualitative changes of oesophageal epithelium using immunohistochemistry with monoclonal antibodies against eosinophil peroxidase and eosinophil major basic protein and mast cell tryptase. Eosinophil infiltration was significantly higher in EO than in GORD both on haematoxylin and eosin staining (54.8 versus 9.1; P < 0.05) and immunohistochemistry (77.5 versus 24.7; P < 0.05). Eosinophil degranulation was significantly more intense in EO than in GORD (1.16 versus 0.41; P < 0.05). Furthermore, eosinophilia-codependent secondary qualitative changes of squamous epithelium in EO were generally more extensive than those in GORD. CONCLUSIONS: Histological differential diagnosis of EO and GORD should be based on eosinophil counts, secondary morphological changes of eosinophils and oesophageal squamous epithelium, especially in cases suspicious of EO.