RESUMO
Developing methods for accurate detection of RNA modifications remains a major challenge in epitranscriptomics. Next-generation sequencing-based mapping approaches have recently emerged but, often, they are not quantitative and lack specificity. Pseudouridine (ψ), produced by uridine isomerization, is one of the most abundant RNA modification. ψ mapping classically involves derivatization with soluble carbodiimide (CMCT), which is prone to variation making this approach only semi-quantitative. Here, we developed 'HydraPsiSeq', a novel quantitative ψ mapping technique relying on specific protection from hydrazine/aniline cleavage. HydraPsiSeq is quantitative because the obtained signal directly reflects pseudouridine level. Furthermore, normalization to natural unmodified RNA and/or to synthetic in vitro transcripts allows absolute measurements of modification levels. HydraPsiSeq requires minute amounts of RNA (as low as 10-50 ng), making it compatible with high-throughput profiling of diverse biological and clinical samples. Exploring the potential of HydraPsiSeq, we profiled human rRNAs, revealing strong variations in pseudouridylation levels at â¼20-25 positions out of total 104 sites. We also observed the dynamics of rRNA pseudouridylation throughout chondrogenic differentiation of human bone marrow stem cells. In conclusion, HydraPsiSeq is a robust approach for the systematic mapping and accurate quantification of pseudouridines in RNAs with applications in disease, aging, development, differentiation and/or stress response.
Assuntos
Pseudouridina/isolamento & purificação , RNA Mensageiro , RNA Ribossômico , RNA de Transferência , Análise de Sequência de RNA/métodos , Células Cultivadas , Humanos , Células-Tronco Mesenquimais , Saccharomyces cerevisiae/genéticaRESUMO
This study investigated the relationship of oxytocin (OT) to chondrogenesis and osteoarthritis (OA). Human bone marrow and multipotent adipose-derived stem cells were cultured in vitro in the absence or presence of OT and assayed for mRNA transcript expression along with histological and immunohistochemical analyses. To study the effects of OT in OA in vivo, a rat model and a human cohort of 63 men and 19 women with hand OA and healthy controls, respectively, were used. The baseline circulating OT, interleukin-6, leptin, and oestradiol levels were measured, and hand X-ray examinations were performed for each subject. OT induced increased aggrecan, collagen (Col) X, and cartilage oligomeric matrix protein mRNA transcript levels in vitro, and the immunolabelling experiments revealed a normalization of Sox9 and Col II protein expression levels. No histological differences in lesion severity were observed between rat OA groups. In the clinical study, a multivariate analysis adjusted for age, body mass index, and leptin levels revealed a significant association between OA and lower levels of OT (odds ratio = 0.77; p = 0.012). Serum OT levels are reduced in patients with hand OA, and OT showed a stimulatory effect on chondrogenesis. Thus, OT may contribute to the pathophysiology of OA.
Assuntos
Condrogênese/efeitos dos fármacos , Osteoartrite/tratamento farmacológico , Ocitocina/farmacologia , Idoso , Animais , Índice de Massa Corporal , Medula Óssea/metabolismo , Técnicas de Cultura de Células , Condrócitos/metabolismo , Colágeno Tipo II/sangue , Estradiol/sangue , Matriz Extracelular/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Interleucina-1beta/metabolismo , Interleucina-6/sangue , Leptina/sangue , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Osteoartrite/metabolismo , Ocitocina/sangue , RNA Mensageiro/metabolismo , Ratos , Fatores de Transcrição SOX9/sangue , Fatores de Transcrição SOX9/metabolismo , Células-Tronco/citologiaRESUMO
Nicorandil is an original vasodilatator used to control angina by decreasing cardiac preload and afterload. Since 1997, many reports of single or multiple nicorandil-induced ulcerations have been published. To date, eight cases of nicorandil-induced fistula into adjacent organs have been described. The pathogeneses of nicorandil-induced ulceration and fistula into adjacent organs are not yet elucidated. The two main hepatic biotransformation pathways of nicorandil are denitration and reduction of the alkyl chain leading to nicotinamide and niconitic acid which merge into the endogenous pool of nicotinamide adenine dinucleotide/phosphate. This merging which is known as saturable, may contribute to a slow and abnormal distribution of nicotinamide and nicotinic acid out of the endogenous pool. Under these special conditions, providing these two molecules in situ, nicotinic acid associated with nicotinamide may ulcerate rather recent or maintained trauma. Ulcers and fistulae induced by nicorandil heal after withdrawal. Surgical intervention is unnecessary and inappropriate as it is ineffective and exacerbates morbidity. All practitioners should be correctly informed about these serious but preventable nicorandil side effects, which mostly occur in the elderly and fragile population. In the absence of corrective measures, withdrawal of this original and active drug should be considered.
Assuntos
Fístula/induzido quimicamente , Nicorandil/efeitos adversos , Nicorandil/farmacocinética , Úlcera/induzido quimicamente , Vasodilatadores/efeitos adversos , Cicatrização , Idoso , Idoso de 80 Anos ou mais , Feminino , Fístula/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Distribuição Tecidual , Úlcera/metabolismo , Vasodilatadores/farmacocinéticaRESUMO
The bioextrusion of mesenchymal stromal cells (MSCs) directly seeded in a bioink enables the production of three-dimensional (3D) constructs, promoting their chondrogenic differentiation. Our study aimed to evaluate the effect of different type I collagen concentrations in the bioink on MSCs' chondrogenic differentiation. We printed 3D constructs using an alginate, gelatin, and fibrinogen-based bioink cellularized with MSCs, with four different quantities of type I collagen addition (0.0, 0.5, 1.0, and 5.0 mg per bioink syringe). We assessed the influence of the bioprinting process, the bioink composition, and the growth factor (TGF-êµ1) on the MSCs' survival rate. We confirmed the biocompatibility of the process and the bioinks' cytocompatibility. We evaluated the chondrogenic effects of TGF-êµ1 and collagen addition on the MSCs' chondrogenic properties through macroscopic observation, shrinking ratio, reverse transcription polymerase chain reaction, glycosaminoglycan synthesis, histology, and type II collagen immunohistochemistry. The bioink containing 0.5 mg of collagen produces the richest hyaline-like extracellular matrix, presenting itself as a promising tool to recreate the superficial layer of hyaline cartilage. The bioink containing 5.0 mg of collagen enhances the synthesis of a calcified matrix, making it a good candidate for mimicking the calcified cartilaginous layer. Type I collagen thus allows the dose-dependent design of specific hyaline cartilage layers.
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Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, plays a key role in the pathogenesis of many inflammatory diseases, including arthritis. Neutralization of this cytokine by anti-TNF-α antibodies has shown its efficacy in rheumatoid arthritis (RA) and is now widely used. Nevertheless, some patients currently treated with anti-TNF-α remain refractory or become nonresponder to these treatments. In this context, there is a need for new or complementary therapeutic strategies. In this study, we investigated in vitro and in vivo anti-inflammatory potentialities of an anti-TNF-α triplex-forming oligonucleotide (TFO), as judged from effects on two rat arthritis models. The inhibitory activity of this TFO on articular cells (synoviocytes and chondrocytes) was verified and compared to that of small interfering RNA (siRNA) in vitro. The use of the anti-TNF-α TFO as a preventive and local treatment in both acute and chronic arthritis models significantly reduced disease development. Furthermore, the TFO efficiently blocked synovitis and cartilage and bone destruction in the joints. The results presented here provide the first evidence that gene targeting by anti-TNF-α TFO modulates arthritis in vivo, thus providing proof-of-concept that it could be used as therapeutic tool for TNF-α-dependent inflammatory disorders.
Assuntos
Artrite/tratamento farmacológico , Autoanticorpos/uso terapêutico , Imunoterapia , Fator de Necrose Tumoral alfa/imunologia , Animais , Artrite/imunologia , Autoanticorpos/imunologia , Células Cultivadas , Modelos Animais de Doenças , RNA Interferente Pequeno/genética , Ratos , Fator de Necrose Tumoral alfa/genéticaRESUMO
Intra-articular (IA) injection of a chondroprotective candidate may delay the osteoarthritis (OA) course, but its rapid absorption into systemic circulation may limit efficacy and produce untoward effects. We compared the pharmacokinetics (PK) of IA rapamycin injected as sustained release in nanoparticles (NPs) versus a free rapamycin suspension in the rat knee compared to an intravenous (IV) free rapamycin shot taken as a reference. Rats received either a single IV injection of free rapamycin (10 µM) or an IA of free or NPs-loaded rapamycin. After sequential exsanguination (15, 30, 60, 180, 360 min, D1, and D7), knee synovial tissue (ST) and cartilage histology were performed. Blood and ST concentrations (LC-MS/MS), PK parameters (area under the curve: AUC; mean residence time: MRT; elimination half-life: T1/2), and IA biocompatibility were assessed. AUCIV was significantly higher for IV than for both IA injections (AUCIA free and AUCIA NPs), with 4248 vs 28 and 74 µg.min.L-1. For ST parameters, we observed a significant difference between AUCIA free and AUCIA NPs with 3735 and 10513 µg.min.L-1 correspondingly. Articular T1/2 and MRT were higher after NPs than after free rapamycin injection: 57.8 and 5.0 h for T1/2 and 80.6 and 5.5 h for MRT, respectively. Histological analysis revealed no chondral injuries and slight transient synovitis only 3 h after the administration of NPs. In the rat knee, rapamycin-loaded NPs delivery via a single IA injection is biocompatible and prolongs synovium joint residency, diminishes blood levels, and reduces detrimental systemic exposure.
Assuntos
Nanopartículas , Sirolimo , Animais , Cromatografia Líquida , Injeções Intra-Articulares , Articulação do Joelho , Ratos , Membrana Sinovial , Espectrometria de Massas em TandemRESUMO
Adult mesenchymal stem cells (MSCs) are currently being investigated as an alternative to chondrocytes for repairing cartilage defects. As several collagen types participate in the formation of cartilage-specific extracellular matrix, we have investigated their gene expression levels during MSC chondrogenic induction. Bone marrow MSCs were cultured in pellet in the presence of BMP-2 and TGF-ß3 for 24 days. After addition of FGF-2, at the fourth passage during MSC expansion, there was an enhancing effect on specific cartilage gene expression when compared to that without FGF-2 at day 12 in pellet culture. A switch in expression from the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of the collagen type II gene was observed at day 24. A short-term addition of FGF-2 followed by a treatment with BMP-2/TGF-ß3 appears sufficient to accelerate chondrogenesis with a particular effect on the main cartilage collagens.
Assuntos
Células da Medula Óssea/metabolismo , Condrogênese/fisiologia , Colágeno/biossíntese , Células-Tronco Mesenquimais/metabolismo , Adulto , Idoso , Células da Medula Óssea/efeitos dos fármacos , Proteína Morfogenética Óssea 2/farmacologia , Proliferação de Células/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Colágeno/genética , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Células-Tronco Mesenquimais/efeitos dos fármacos , Pessoa de Meia-Idade , Isoformas de Proteínas , Fator de Crescimento Transformador beta3/farmacologiaRESUMO
Osteoarthritis (OA) is the most common degenerative joint disease. Rapamycin is a potential candidate for OA treatment by increasing the autophagy process implicated in its physiopathology. To optimize Rapamycin profit and avoid systemic side effects, intra-articular (i.a.) administration appeared helpful. However, Rapamycin's highly hydrophobic nature and low bioavailability made it challenging to develop purpose-made drug delivery systems to overcome these limitations. We developed Rapamycin-loaded nanoparticles (NPs) using poly (lactic-co-glycolic acid) by emulsion/evaporation method. We evaluated these NPs' cytocompatibility towards cartilage (chondrocytes) and synovial membrane cells (synoviocytes) for a potential i.a. administration. The in vitro characterization of Rapamycin-loaded NPs had shown a suitable profile for an i.a. administration. In vitro biocompatibility of NPs was highlighted to 10 µM of Rapamycin for both synoviocytes and chondrocytes, but significant toxicity was observed with higher concentrations. Besides, synoviocytes are more sensitive to Rapamycin-loaded NPs than chondrocytes. Finally, we observed in vitro that an adapted formulated Rapamycin-loaded NPs could be safe at suitable i.a. injection concentrations. The toxic effect of Rapamycin encapsulated in these NPs on both articular cells was dose-dependent. After Rapamycin-loaded NPs i.a. administration, local retention, in situ safety, and systemic release should be evaluated with experimental in vivo models.
Assuntos
Nanopartículas , Sirolimo , Portadores de Fármacos , Glicóis , Injeções Intra-Articulares , Nanopartículas/toxicidade , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Sirolimo/toxicidadeRESUMO
To evaluate whether the risk of bone fragility on computed tomography (CT) (scanographic bone attenuation coefficient of the first lumbar vertebra (SBAC-L1)) is associated with the severity of spine structural involvement (mSASSS) in patients with ankylosing spondylitis (AS). This retrospective study included AS patients, followed from 2009 to 2017, who fulfilled the New York criteria and who underwent thoraco-abdomino-pelvic CT and radiography (spine, pelvis). The structural involvement was retained for mSASSS ≥ 2. The SBAC-L1 was measured in Hounsfield units (HU). A SBAC-L1 ≤ 145 HU was used to define patients at risk of vertebral fracture (VF). A total of 73 AS patients were included (mean age: 60.3 (± 10.7) years, 8 women (11%), mean disease duration: 24.6 years (± 13.9)). Sixty patients (82.2%) had a mSASSS ≥ 2 (mean score 20.7 (± 21.2)). The mean SBAC-L1 was 141.1 HU (± 45), 138.1 HU (± 44.8) and 154.8 HU (± 44.9) in the total, mSASSS ≥ 2 and mSASSS < 2 populations, respectively. Patients with bone bridges had lower SBAC-L1 than mSASSS ≥ 2 patients without ankylosis (p = 0.02) and more often SBAC-L1 ≤ 145 HU (73% vs 41.9%, p = 0.006). A SBAC-L1 ≤ 145 HU was not associated with structural spine involvement, but patients with bone bridges had significantly decreased SBAC-L1 and an increased probability of being under the fracture threshold.
Assuntos
Vértebras Lombares/diagnóstico por imagem , Fraturas da Coluna Vertebral/diagnóstico por imagem , Espondilite Anquilosante/diagnóstico por imagem , Tomografia Computadorizada por Raios X , Idoso , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Índice de Gravidade de DoençaRESUMO
OBJECTIVE: To assess OA-related changes in mean compartmental femorotibial cartilage thickness in rat knees by three-dimensional (3D) MRI (7T). METHODS: MRI was performed in vivo at 7T on OA and untouched contralateral knee joints. Gradient Echo Fast Imaging 3D MR images were acquired sequentially in surgically induced OA (D0) in 40 Wistar rats (anterior cruciate ligament transection). Mean femoral (trochlear, lateral and medial) and tibial (lateral and medial) cartilage thicknesses were quantified from a 2D MRI slide in weight-bearing areas and from a 3D MRI data set. At each time-point [Day (D)8, D14, D21, D40 and D60], eight animals (16 knees) were sacrificed for concomitant histomorphometry. RESULTS: As body weight dramatically increased throughout the experiment (+150%, baseline vs endpoint), all compartmental mean cartilage thicknesses noticeably decreased (D8, D14) and then remained relatively stable. Femoral compartments in OA knees were thinner at the end of the experiment than in contralateral age-matched knees. Conversely, lateral and medial tibial cartilages were thicker than controls. Histological correlation was significant only in untouched healthy cartilages (3D better than 2D). CONCLUSIONS: 3D MRI (7T) enables in vivo monitoring of compartmental changes in OA-related femorotibial rat cartilage thickness vs contralateral age-matched knees.
Assuntos
Ligamento Cruzado Anterior/metabolismo , Artrite Experimental/patologia , Cartilagem Articular/metabolismo , Osteoartrite do Joelho/patologia , Análise de Variância , Animais , Estudos Transversais , Modelos Animais de Doenças , Fêmur , Imageamento Tridimensional , Articulação do Joelho , Imageamento por Ressonância Magnética/métodos , Masculino , Ratos , Ratos Wistar , TíbiaRESUMO
Hyaline cartilage is deficient in self-healing properties. The early treatment of focal cartilage lesions is a public health challenge to prevent long-term degradation and the occurrence of osteoarthritis. Cartilage tissue engineering represents a promising alternative to the current insufficient surgical solutions. 3D printing is a thriving technology and offers new possibilities for personalized regenerative medicine. Extrusion-based processes permit the deposition of cell-seeded bioinks, in a layer-by-layer manner, allowing mimicry of the native zonal organization of hyaline cartilage. Mesenchymal stem cells (MSCs) are a promising cell source for cartilage tissue engineering. Originally isolated from bone marrow, they can now be derived from many different cell sources (e.g., synovium, dental pulp, Wharton's jelly). Their proliferation and differentiation potential are well characterized, and they possess good chondrogenic potential, making them appropriate candidates for cartilage reconstruction. This review summarizes the different sources, origins, and densities of MSCs used in extrusion-based bioprinting (EBB) processes, as alternatives to chondrocytes. The different bioink constituents and their advantages for producing substitutes mimicking healthy hyaline cartilage is also discussed.
Assuntos
Bioimpressão/métodos , Células-Tronco Mesenquimais/citologia , Osteoartrite/terapia , Impressão Tridimensional , Engenharia Tecidual/métodos , Alicerces Teciduais , Alginatos/uso terapêutico , Animais , Cartilagem Articular/citologia , Humanos , Cartilagem Hialina/citologia , Hidrogéis/uso terapêuticoRESUMO
INTRODUCTION: Ankylosing spondylitis (AS) patients seems to be at risk of osteoporosis but bone screening is not often performed. The objective was to evaluate the effect of vertebral ankylosis on scanographic bone attenuation coefficient (SBAC) on lumbar vertebrae in AS patients. METHODS: This study included AS patients fulfilling New York criteria who underwent both thoraco-abdomino-pelvic computed tomography and X-rays during routine follow-up. The modified stoke ankylosing spondylitis spinal score (mSASSS) was scored on X-rays, and the presence of at least one syndesmophyte (mSASSS≥2) defined mSASSS+ patients. Ankylosis of a lumbar vertebra was defined by the presence of bone bridges to its two adjacent vertebrae. The SBAC was measured from L1 to L5, and the fracture threshold was set at SBAC≤145 HU. RESULTS: A total of 73 AS patients were included (mean age: 60.3 [±10.7] years, 65 men [89%]). Sixty patients (82.2%) were mSASSS+; 13 patients (17.8%) presented ankylosis of at least one lumbar vertebra. The SBAC of each lumbar vertebra was not significantly different between mSASSS- and mSASSS+ patients. The SBAC was lower for patients with at least one bone bridge than for patients without (P<0.05). Patients with lumbar vertebral ankylosis had a higher risk of presenting an SBAC≤145 HU (OR: 4.95 (95% CI: 1.1-17.4)). CONCLUSION: The presence of a bone bridge and complete ankylosis of lumbar vertebra were associated with a higher risk of SBAC under the fracture threshold, suggesting structural deterioration of trabecular bone in ankylosed vertebrae in AS patients.
Assuntos
Osteoporose , Espondilite Anquilosante , Densidade Óssea , Humanos , Vértebras Lombares/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Espondilite Anquilosante/complicações , Espondilite Anquilosante/diagnóstico por imagem , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: MSCs isolated from bone marrow (BM-MSCs) have well-established chondrogenic potential, but MSCs derived from the synovial membrane (SM-MSCs) and synovial fluid (SF-MSCs) are thought to possess superior chondrogenicity. This study aimed to compare the in vitro immunophenotype and trilineage and chondrogenic potential of BM-MSCs to SM-MSCs and SF-MSCs. METHODS: MSCs were isolated from bone marrow (BM-MSCs), synovial membrane (SM-MSCs), and synovial fluid (SF-MSCs) extracted from the hips (BM) and knees (SM and SF) of advanced OA patients undergoing arthroplasty. Flow cytometric analysis was used at P2 to evaluate cell stemness. The trilinear differentiation test was performed at P2. At P3, MSC-seeded collagen sponges were cultured in chondrogenic medium for 28 days. Chondrogenic gene expression was quantified by qRT-PCR. Finally, the implants were stained to assess the deposition of proteoglycans and type II collagen. RESULTS: Despite variability, the immunophenotyping of BM-MSCs, SM-MSCs, and SF-MSCs was quite similar. All cell types were positive for the expression of stem cell markers and negative for exclusion markers. Additionally, chondrogenic differentiation and hypertrophy were more pronounced in BM-MSCs (ACAN, SOX9, COL2B, and COL10A) than in SF-MSCs, with SM-MSCs having intermediate characteristics. Concerning matrix synthesis, the three cell types were equipotent in terms of GAG content, while BM-MSC ECM synthesis of type II collagen was superior. CONCLUSIONS: Chondrogenic MSCs are easily collected from SM and SF in advanced human OA, but in vitro chondrogenesis that is superior to age-matched BM-MSCs should not be expected. However, due to intra-articular priming, SF-MSCs did not overexpress hypertrophic gene.
Assuntos
Condrogênese , Células-Tronco Mesenquimais , Medula Óssea , Células da Medula Óssea , Diferenciação Celular , Células Cultivadas , Humanos , Líquido Sinovial , Membrana SinovialRESUMO
3D bioprinting offers interesting opportunities for 3D tissue printing by providing living cells with appropriate scaffolds with a dedicated structure. Biological advances in bioinks are currently promising for cell encapsulation, particularly that of mesenchymal stem cells (MSCs). We present herein the development of cartilage implants by 3D bioprinting that deliver MSCs encapsulated in an original bioink at low concentration. 3D-bioprinted constructs (10 × 10 × 4 mm) were printed using alginate/gelatin/fibrinogen bioink mixed with human bone marrow MSCs. The influence of the bioprinting process and chondrogenic differentiation on MSC metabolism, gene profiles, and extracellular matrix (ECM) production at two different MSC concentrations (1 million or 2 million cells/mL) was assessed on day 28 (D28) by using MTT tests, real-time RT-PCR, and histology and immunohistochemistry, respectively. Then, the effect of the environment (growth factors such as TGF-ß1/3 and/or BMP2 and oxygen tension) on chondrogenicity was evaluated at a 1 M cell/mL concentration on D28 and D56 by measuring mitochondrial activity, chondrogenic gene expression, and the quality of cartilaginous matrix synthesis. We confirmed the safety of bioextrusion and gelation at concentrations of 1 million and 2 million MSC/mL in terms of cellular metabolism. The chondrogenic effect of TGF-ß1 was verified within the substitute on D28 by measuring chondrogenic gene expression and ECM synthesis (glycosaminoglycans and type II collagen) on D28. The 1 M concentration represented the best compromise. We then evaluated the influence of various environmental factors on the substitutes on D28 (differentiation) and D56 (synthesis). Chondrogenic gene expression was maximal on D28 under the influence of TGF-ß1 or TGF-ß3 either alone or in combination with BMP-2. Hypoxia suppressed the expression of hypertrophic and osteogenic genes. ECM synthesis was maximal on D56 for both glycosaminoglycans and type II collagen, particularly in the presence of a combination of TGF-ß1 and BMP-2. Continuous hypoxia did not influence matrix synthesis but significantly reduced the appearance of microcalcifications within the extracellular matrix. The described strategy is very promising for 3D bioprinting by the bioextrusion of an original bioink containing a low concentration of MSCs followed by the culture of the substitutes in hypoxic conditions under the combined influence of TGF-ß1 and BMP-2.
RESUMO
Both chondrocytes and mensenchymal stem cells (MSCs) are the most used cell sources for cartilage tissue engineering. However, monolayer expansion to obtain sufficient cells leads to a rapid chondrocyte dedifferentiation and a subsequent ancillary reduced ability of MSCs to differentiate into chondrocytes, thus limiting their application in cartilage repair. The aim of this study was to investigate the influence of the monolayer expansion on the immunophenotype and the gene expression profile of both cell types, and to find the appropriate compromise between monolayer expansion and the remaining chondrogenic characteristics. To this end, human chondrocytes, isolated enzymatically from femoral head slice, and human MSCs, derived from bone marrow, were maintained in monolayer culture up to passage 5. The respective expressions of cell surface markers (CD34, CD45, CD73, CD90, CD105, CD166) and several chondrogenic-related genes for each passage (P0-P5) of those cells were then analyzed using flow cytometry and quantitative real-time PCR, respectively. Flow cytometry analyses showed that, during the monolayer expansion, some qualitative and quantitative regulations occur for the expression of cell surface markers. A rapid increase in mRNA expression of type 1 collagen occurs whereas a significant decrease of type 2 collagen and Sox 9 was observed in chondrocytes through the successive passages. On the other hand, the expansion did not induced obvious change in MSCs gene expression. In conclusion, our results suggest that passage 1 might be the up-limit for chondrocytes in order to achieve their subsequent redifferentiation in 3D scaffold. Nevertheless, MSCs could be expanded in monolayer until passage 5 without loosing their undifferentiated phenotypes.
Assuntos
Antígenos CD/metabolismo , Biomarcadores/metabolismo , Condrócitos/metabolismo , Expressão Gênica , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Idoso , Idoso de 80 Anos ou mais , Medula Óssea , Técnicas de Cultura de Células/métodos , Desdiferenciação Celular/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Condrócitos/citologia , Condrogênese/fisiologia , Colágeno/metabolismo , Citometria de Fluxo/métodos , Humanos , Pessoa de Meia-Idade , Fatores de Transcrição SOX9/metabolismo , Engenharia Tecidual/métodosRESUMO
AIM: to determine if chondrocytic Hsp70 induction, via intra-articular injections of a reversible proteasome inhibitor (MG132), can protect articular chondrocytes from cellular death in experimental rat OA knee induced surgically by anterior cruciate ligament transection (ACLT). MATERIALS AND METHODS: ACLT was performed on D0. Histological lesions in naive (sham) controls (ACLT+saline) and treated (ACLT+MG132) rats were assessed according to Mankin's score. Repeated intra-articular injections (1.5 muM MG132 or saline were performed on D1, D7, D14 and D21. Rats were sacrificed sequentially on D7, D14 and D28. Detection of active caspase-3 and protein expression of Hsp70 was also determined on D7, D14 and D28 by immunostaining methods. RESULTS: MG132 significantly reduced OA lesions on D28 in the MG132 treated group. The expression of Hsp70 increased 11-fold in the MG132-treated group versus 2-3-fold in ACLT-control rats on D28. Concomitantly, cells expressing caspase-3 increased 4-fold in ACLT model and decreased 2-fold with MG132 treatment. CONCLUSIONS: Intra-articular induction of Hsp70 by MG132 could be a safe and interesting tool in chondrocytes protection from cellular injuries and thus might be a novel chondroprotective modality in rat OA.
Assuntos
Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Modelos Animais de Doenças , Proteínas de Choque Térmico HSP70/metabolismo , Leupeptinas/administração & dosagem , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/prevenção & controle , Inibidores de Proteassoma , Animais , Artroplastia/efeitos adversos , Inibidores de Cisteína Proteinase/administração & dosagem , Masculino , Osteoartrite do Joelho/etiologia , Osteoartrite do Joelho/patologia , Ratos , Ratos Wistar , Resultado do TratamentoRESUMO
OBJECTIVES: to assess reliability and reproducibility of quantitative MRI (7 T) in assessing rat femoro-tibial cartilage volume. METHODS: 5 healthy rat knees were scanned in vivo using a 7 T experimental imager. Sagittal high resolution 3D Gradient Echo with fat suppression sequences were performed with a dedicated home-made 2-elements array coil. 3D MRI sets were used to perform manual segmentation of the 3 cartilage compartments (femoral groove, medial and lateral tibial plateaus) by using a tactile screen. To evaluate inter- and intra-observer reproducibilities, the segmentation procedure was done blindly by two trained observers. One observer repeated the operation twice, with a period of 10 months between both readings. RESULTS: the mean duration to manually segment all the slices covering the cartilaginous joint was 4 hours. On the one hand, the inter-observer root mean square of coefficients of variation was 9.1%, 6.2%, 9.6% for the femoral, medial and lateral tibial compartments respectively. On the other hand, the intra-observer reproducibility was 2.1%, 3.2%, 2.5% for these cartilage compartments cited above. CONCLUSION: the image quality obtained at 7 Teslas with our dedicated coil allowed segmentation of the cartilage compartments with good reproducibility. This study demonstrated that MRI is a useful technology to provide a non-invasive and reliable assessment of rat knee cartilage volume.
Assuntos
Cartilagem Articular/anatomia & histologia , Aumento da Imagem/instrumentação , Imageamento Tridimensional/instrumentação , Articulação do Joelho/anatomia & histologia , Imageamento por Ressonância Magnética/instrumentação , Animais , Desenho de Equipamento , Análise de Falha de Equipamento , Estudos de Viabilidade , Imageamento Tridimensional/métodos , Imageamento por Ressonância Magnética/métodos , Masculino , Tamanho do Órgão , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The purpose of this study was to assess 2 Gd-based macromolecular intravascular contrast agents (P792, rapid clearance blood pool agent (rBPA) and P717, slow clearance blood pool agent (sBPA)) compared to Gd-DOTA (representative extracellular non specific agent) in MR imaging of knee rabbit experimental synovitis. Quantitative dynamic contrast enhanced MRI (qDCE-MRI) after intravascular injection of a low molecular weight contrast agent of 0.56 kDa (Gd-DOTA) and 2 high-molecular-weight contrast agents of 6.47 kDa (P792) and 52 kDa (P717) was performed in rabbits with carrageenan-induced synovitis of the right knee. P792 and P717 provided a progressive and persistent enhancement of arthritic synovial tissue while Gd-DOTA provided an early and rapidly declining enhancement with a concomitant diffusion in synovial fluid, thus limitating delineation of synovial pannus. P792 allowed acquisition of high-quality MR arthrograms, due to both a better diffusion in synovial pannus (vs. P717) and a concomitant restricted diffusion into the synovial fluid (vs. Gd-DOTA). In fact, experimental rabbit synovitis represent a specific entity that favors the T1 effect of high-molecular-weight agents, and especially rBPA P792, entrapped in synovial pannus, without diffusion in the synovial fluid. Due to this lack of arthrographic effect, P792 accumulation could be specifically sequentially analyzed by qDCE-MRI for detecting, characterizing and monitoring synovial vascular permeability changes during mono- or polysynovitis.
Assuntos
Modelos Animais de Doenças , Compostos Heterocíclicos , Aumento da Imagem/métodos , Articulação do Joelho/patologia , Compostos Organometálicos , Sinovite/diagnóstico , Animais , Meios de Contraste , Imageamento por Ressonância Magnética , Masculino , Coelhos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Cartilage tissue engineering is making progress, but the competing available strategies still leave room for improvement and consensual overviews regarding the best combinations of scaffolds and cell sources are limited by the capacity to compare them directly. In addition, because most strategies involve autologous cell transfer, once these are optimized, the resulting implants require individual quality control prior to grafting in order to emphasize patient-to-patient differential responsiveness to engineering processes. Here, cartilage substitutes prepared from human mesenchymal stem cells undergoing chondrogenic differentiation within distinct scaffolds were used as pilot samples to investigate the pertinence of a novel method with the aim of characterizing the implants. The limits and advantages of analysing, by label-free liquid chromatography-coupled matrix-assisted laser desorption and ionization (LC-MALDI) mass spectrometry, the secreted proteome released into culture medium by engineered cartilage tissues were investigated and compared with more classically used methods for biomaterial characterization. This method did not require sacrificing the biomaterials and robustly evidenced their chondrogenic statuses. In more detail, the method highlighted differences between batches prepared from distinct donors. It was adapted to distinct scaffolds and allowed a comparison of the influence of individual engineering steps, such as growth factor combinations and oxygen tension. Finally, it evidenced subtle changes between replicate substitutes within a series, thereby distinguishing the least and most accomplished ones. We conclude that relative quantification of secreted proteins through label-free LC-MALDI will be useful, not only to orientate engineering methodologies, but also to ultimately provide non-invasive quality control of engineered tissue substitutes for the repair of cartilage and possibly other connective tissues.
Assuntos
Bioengenharia , Cartilagem Articular/patologia , Condrogênese , Proteômica/métodos , Regeneração , Alicerces Teciduais/química , Idoso , Idoso de 80 Anos ou mais , Materiais Biocompatíveis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Células Cultivadas , Microambiente Celular/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Humanos , Implantes Experimentais , Pessoa de Meia-Idade , Proteoma/metabolismo , Controle de Qualidade , Regeneração/efeitos dos fármacos , Coloração e Rotulagem , Doadores de TecidosRESUMO
BACKGROUND: Mesenchymal stem cells (MSCs) are found in synovial fluid (SF) and can easily be harvested during arthrocentesis or arthroscopy. However, SF-MSC characterization and chondrogenicity in collagen sponges have been poorly documented as well as their hypothetical in vivo chondroprotective properties with intra-articular injections during experimental osteoarthritis (OA). METHODS: SF-MSCs were isolated from human SF aspirates in patients suffering from advanced OA undergoing total knee joint replacements. SF-MSCs at passage 2 (P2) were characterized by flow cytometry for epitope profiling. SF-MSCs at P2 were subsequently cultured in vitro to assess their multilineage potentials. To assess their chondrogenicity, SF-MSCs at P4 were seeded in collagen sponges for 4 weeks under various oxygen tensions and growth factors combinations to estimate their gene profile and matrix production. Also, SF-MSCs were injected into the joints in a nude rat anterior cruciate ligament transection (ACLT) to macroscopically and histologically assess their possible chondroprotective properties,. RESULTS: We characterized the stemness (CD73+, CD90+, CD105+, CD34-, CD45-) and demonstrated the multilineage potency of SF-MSCs in vitro. Furthermore, the chondrogenic induction (TGF-ß1 ± BMP-2) of these SF-MSCs in collagen sponges demonstrated a good capacity of chondrogenic gene induction and extracellular matrix synthesis. Surprisingly, hypoxia did not enhance matrix synthesis, although it boosted chondrogenic gene expression (ACAN, SOX9, COL2A1). Besides, intra-articular injections of xenogenic SF-MSCs did exert neither chondroprotection nor inflammation in ACLT-induced OA in the rat knee. CONCLUSIONS: Advanced OA SF-MSCs seem better candidates for cell-based constructs conceived for cartilage defects rather than intra-articular injections for diffuse OA.