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1.
Allergy ; 77(7): 2025-2037, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35094406

RESUMEN

Regulatory non-coding RNAs (ncRNAs) including small non-coding RNAs (sRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) have gained considerable attention in the last few years. This is mainly due to their condition- and tissue-specific expression and their various modes of action, which suggests them as promising biomarkers and therapeutic targets. One important mechanism of ncRNAs to regulate gene expression is through translation of short open reading frames (sORFs). These sORFs can be located in lncRNAs, in non-translated regions of mRNAs where upstream ORFs (uORFs) represent the majority, or in circRNAs. Regulation of their translation can function as a quick way to adapt protein production to changing cellular or environmental cues, and can either depend solely on the initiation and elongation of translation, or on the roles of the produced functional peptides. Due to the experimental challenges to pinpoint translation events and to detect the produced peptides, translational regulation through regulatory RNAs is not well studied yet. In the case of circRNAs, they have only recently started to be recognized as regulatory molecules instead of mere artifacts of RNA biosynthesis. Of the many roles described for regulatory ncRNAs, we will focus here on their regulation during inflammation and in immunity.


Asunto(s)
ARN Largo no Codificante , Humanos , Inflamación/genética , Péptidos , Biosíntesis de Proteínas , ARN Circular , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo
2.
Int J Mol Sci ; 22(9)2021 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-33946412

RESUMEN

Despite the huge body of research on osteogenic differentiation and bone tissue engineering, the translation potential of in vitro results still does not match the effort employed. One reason might be that the protocols used for in vitro research have inherent pitfalls. The synthetic glucocorticoid dexamethasone is commonly used in protocols for trilineage differentiation of human bone marrow mesenchymal stromal cells (hBMSCs). However, in the case of osteogenic commitment, dexamethasone has the main pitfall of inhibiting terminal osteoblast differentiation, and its pro-adipogenic effect is well known. In this work, we aimed to clarify the role of dexamethasone in the osteogenesis of hBMSCs, with a particular focus on off-target differentiation. The results showed that dexamethasone does induce osteogenic differentiation by inhibiting SOX9 expression, but not directly through RUNX2 upregulation as it is commonly thought. Rather, PPARG is concomitantly and strongly upregulated, leading to the formation of adipocyte-like cells within osteogenic cultures. Limiting the exposure to dexamethasone to the first week of differentiation did not affect the mineralization potential. Gene expression levels of RUNX2, SOX9, and PPARG were simulated using approximate Bayesian computation based on a simplified theoretical model, which was able to reproduce the observed experimental trends but with a different range of responses, indicating that other factors should be integrated to fully understand how dexamethasone influences cell fate. In summary, this work provides evidence that current in vitro differentiation protocols based on dexamethasone do not represent a good model, and further research is warranted in this field.


Asunto(s)
Dexametasona/farmacología , Glucocorticoides/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Osteogénesis/efectos de los fármacos , PPAR gamma/metabolismo , Factor de Transcripción SOX9/metabolismo , Adulto , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , PPAR gamma/genética , Factor de Transcripción SOX9/genética
3.
Calcif Tissue Int ; 107(4): 301-318, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32710266

RESUMEN

Mechanotransduction is pivotal in the maintenance of homeostasis in different tissues and involves multiple cell signaling pathways. In bone, mechanical stimuli regulate the balance between bone formation and resorption; osteocytes play a central role in this regulation. Dysfunctions in mechanotransduction signaling or in osteocytes response lead to an imbalance in bone homeostasis. This alteration is very relevant in some conditions such as osteoporosis and aging. Both are characterized by increased bone weakness due to different causes, for example, the increase of osteocyte apoptosis that cause an alteration of fluid space, or the alteration of molecular pathways. There are intertwined yet very different mechanisms involved among the cell-intrinsic effects of aging on bone, the cell-intrinsic and tissue-level effects of estrogen/androgen withdrawal on bone, and the effects of reduced mechanical loading on bone, which are all involved to some degree in how aged bone fails to respond properly to stress/strain compared to younger bone. This review aims at clarifying how the cellular and molecular pathways regulated and induced in bone by mechanical stimulation are altered with aging and in osteoporosis, to highlight new possible targets for antiresorptive or anabolic bone therapeutic approaches.


Asunto(s)
Envejecimiento , Huesos/fisiología , Osteoporosis/patología , Soporte de Peso , Anciano , Huesos/fisiopatología , Humanos , Mecanotransducción Celular , Osteocitos , Estrés Mecánico
4.
Int J Mol Sci ; 21(3)2020 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-32019244

RESUMEN

As the population of western societies on average ages, the number of people affected by bone remodeling-associated diseases such as osteoporosis continues to increase. The development of new therapeutics is hampered by the high failure rates of drug candidates during clinical testing, which is in part due to the poor predictive character of animal models during preclinical drug testing. Co-culture models of osteoblasts and osteoclasts offer an alternative to animal testing and are considered to have the potential to improve drug development processes in the future. However, a robust, scalable, and reproducible 3D model combining osteoblasts and osteoclasts for preclinical drug testing purposes has not been developed to date. Here we review various types of osteoblast-osteoclast co-culture models and outline the remaining obstacles that must be overcome for their successful translation.


Asunto(s)
Conservadores de la Densidad Ósea/farmacología , Remodelación Ósea/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Osteoblastos/citología , Osteoclastos/citología , Osteoporosis/tratamiento farmacológico , Animales , Técnicas de Cocultivo , Humanos , Osteoblastos/efectos de los fármacos , Osteoclastos/efectos de los fármacos
5.
Molecules ; 25(19)2020 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-33019648

RESUMEN

This bibliometric review aimed to identify and analyze the top 100 most-cited publications on the systemic manifestations of periodontal disease (PD). A literature search was performed using the Web of Science (WoS) 'All Databases', without any restriction of language, publication year, or study design. Of 4418 articles, the top 100 were included based on their citation count. After downloading the full texts, their bibliometric information was extracted and analyzed. The citation counts for the top 100 articles ranged from 156 to 4191 (median 217). The most productive years were 2003 and 2005, with 20 articles on the list. Majority of the articles were published in the Journal of Periodontology (n = 25). The top 100 articles were generated primarily from the USA (n = 61). Most of the publications were clinical trials (n = 27) and focused on the cardiovascular manifestations of PD (n = 31). Most of the articles were within the evidence level V (n = 41). A total of 58 studies received funding and the most frequently used keyword in the top articles was "periodontal disease" (n = 39). The current citation analysis presents insights into the current trends in the systemic manifestations of periodontal disease.


Asunto(s)
Bibliometría , Enfermedades Periodontales/patología , Animales , Autoria , Humanos , Publicaciones
6.
J Cell Physiol ; 233(6): 4401-4407, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29206301

RESUMEN

The isolation of good quality RNA from tissues is an essential prerequisite for gene expression analysis to study pathophysiological processes. This study evaluated the RNA isolated from human trabecular bone and defined a set of stable reference genes. After pulverization, RNA was extracted with a phenol/chloroform method and then purified using silica columns. The A260/280 ratio, A260/230 ratio, RIN, and ribosomal ratio were measured to evaluate RNA quality and integrity. Moreover, the expression of six candidates was analyzed by qPCR and different algorithms were applied to assess reference gene stability. A good purity and quality of RNA was achieved according to A260/280 and A260/230 ratios, and RIN values. TBP, YWHAZ, and PGK1 were the most stable reference genes that should be used for gene expression analysis. In summary, the method proposed is suitable for gene expression evaluation in human bone and a set of reliable reference genes has been identified.


Asunto(s)
Proteínas 14-3-3/genética , Hueso Esponjoso/química , Fosfoglicerato Quinasa/genética , ARN/genética , Proteína de Unión a TATA-Box/genética , Perfilación de la Expresión Génica/normas , Marcadores Genéticos , Humanos , ARN/aislamiento & purificación , Estabilidad del ARN , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Estándares de Referencia , Reproducibilidad de los Resultados
7.
J Cell Physiol ; 232(8): 2201-2209, 2017 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-27976811

RESUMEN

Chronic Kidney Disease-Mineral and Bone Disorder (CKD-MBD) is characterized by an increased fracture risk. Bone marrow mesenchymal stromal cells (BMSCs) may be involved in the pathogenesis of bone disease and, in view of their promising potential applications in bone tissue engineering, the effect of uremia on BMSCs regenerative potential represents a central issue. The present study evaluated in vitro the effect of a serum pool from hemodialysis patients on BMSCs to observe its influence on osteogenic differentiation. Besides alterations in spatial organization and cytotoxicity along with hyperproliferation, gene expression analysis suggested an impairment in the osteogenic differentiation. More importantly, Receptor activator of nuclear factor kappa-B ligand (RANKL) was upregulated with a mild reduction in osteoprotegerin levels. In summary, uremic environment seems to impair BMSCs osteogenic differentiation. Moreover BMSCs themselves may enhance osteoclastogenesis, feasibly contributing to the altered bone remodeling in CKD-MBD patients. J. Cell. Physiol. 232: 2201-2209, 2017. © 2016 Wiley Periodicals, Inc.


Asunto(s)
Células de la Médula Ósea/metabolismo , Diferenciación Celular , Fallo Renal Crónico/sangre , Células Madre Mesenquimatosas/metabolismo , Osteogénesis , Uremia/sangre , Anciano , Anciano de 80 o más Años , Fosfatasa Alcalina/metabolismo , Apoptosis , Células de la Médula Ósea/patología , Puntos de Control del Ciclo Celular , Linaje de la Célula , Proliferación Celular , Forma de la Célula , Supervivencia Celular , Células Cultivadas , Microambiente Celular , Femenino , Regulación de la Expresión Génica , Humanos , Fallo Renal Crónico/genética , Fallo Renal Crónico/patología , Fallo Renal Crónico/terapia , Masculino , Células Madre Mesenquimatosas/patología , Persona de Mediana Edad , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Fenotipo , Ligando RANK/genética , Ligando RANK/metabolismo , Diálisis Renal , Factores de Tiempo , Uremia/genética , Uremia/patología , Uremia/terapia
8.
Cytotherapy ; 18(5): 593-612, 2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-27059198

RESUMEN

Osteoarthritis (OA) can affect every joint, especially the knee. Given the complexity of this pathology, OA is difficult to treat with current therapies, which only relieve pain and inflammation and are not capable of restoring tissues once OA has started. Currently, researchers focus on finding a therapeutic strategy that may help to arrest disease progression. The present narrative review gives an overview of the genes involved in the development and progression of OA, assessing in vivo studies performed in knock-out mice affected by OA, to suggest new therapeutic strategies. The article search was performed on the PubMed database and www.webofknowledge.com website with the following keywords: "knee osteoarthritis" AND "knockout mice". The included studies were in English and published from 2005 to 2015. Additional papers were found within the references of the selected articles. In the 55 analyzed in vivo studies, genes mainly affected chondrocyte homeostasis, inflammatory processes, extracellular matrix and the relationship between obesity and OA. Genes are defined as inducing, preventing and not influencing OA. This review shows that joint homeostasis depends on a variety of genetic factors, and preventing or restoring the loss of a gene encoding for protective proteins, or inhibiting the expression of proteins that induce OA, might be a potential therapeutic approach. However, conclusions cannot be drawn because of the wide variability concerning the technique used for OA induction, the role of the genes, the method for tissue evaluations and the lack of assessments of all joint tissues.


Asunto(s)
Condrocitos/metabolismo , Progresión de la Enfermedad , Osteoartritis de la Rodilla/genética , Osteoartritis de la Rodilla/terapia , Animales , Modelos Animales de Enfermedad , Matriz Extracelular/genética , Inflamación/genética , Inflamación/inmunología , Masculino , Ratones , Ratones Noqueados , Obesidad/complicaciones , Osteoartritis de la Rodilla/complicaciones
9.
Cytotherapy ; 17(11): 1536-44, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26305076

RESUMEN

BACKGROUND AIMS: Aging and estrogen deficiency play a pivotal role in reducing tenocyte proliferation, collagen turnover and extracellular matrix remodeling. Mesenchymal stromal cells are being studied as an alternative for tendon regeneration, but little is known about the molecular events of adipose-derived mesenchymal stromal cells (ADSCs) on tenocytes in tendons compromised by aging and estrogen deficiency. The present in vitro study aims to compare the potential therapeutic effects of ADSCs, harvested from healthy young (sham) and aged estrogen-deficient (OVX) subjects, for tendon healing. METHODS: An indirect co-culture system was set up with ADSCs, isolated from OVX or sham rats, and tenocytes from OVX rats. Cell proliferation, healing rate and gene expression were evaluated in both a standard culture condition and a microwound-healing model. RESULTS: It was observed that tenocyte proliferation, healing rate and collagen expression improved after the addition of sham ADSCs in both culture situations. OVX ADSCs also increased tenocyte proliferation and healing rate but less compared with sham ADSCs. Decorin and Tenascin C expression increased in the presence of OVX ADSCs. CONCLUSIONS: Findings suggest that ADSCs might be a promising treatment for tendon regeneration in advanced age and estrogen deficiency. However, some differences between allogenic and autologous cells were found and should be investigated in further in vivo studies. It appears that allogenic ADSCs improve tenocyte proliferation, collagen expression and the healing rate more than autologous cells. Autologous cells increase collagen expression only in the absence of an injury and increase Decorin and Tenascin C more than allogenic cells.


Asunto(s)
Tejido Adiposo/citología , Envejecimiento/fisiología , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Estrógenos/metabolismo , Células Madre Mesenquimatosas/fisiología , Tendones/patología , Envejecimiento/metabolismo , Animales , Proliferación Celular , Técnicas de Cocultivo , Colágeno/genética , Colágeno/metabolismo , Decorina/genética , Decorina/metabolismo , Matriz Extracelular/metabolismo , Femenino , Expresión Génica , Células Madre Mesenquimatosas/citología , Ovariectomía , Ratas Sprague-Dawley , Tenascina/genética , Tenascina/metabolismo , Tendones/citología
10.
Cytotherapy ; 17(2): 215-23, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25484309

RESUMEN

BACKGROUND AIMS: Tendon is a complex tissue with a reduced regenerative ability. Nowadays, little or nothing is known about the regenerative effect of adipose-derived mesenchymal stromal cells (ADSCs) on tendons. METHODS: The study aimed to evaluate the in vitro mutual interaction of ADSCs and tenocytes in standard culture conditions and a microwound healing model. Tenocyte viability, microwound recovery and the expression of genes encoding for the main extracellular matrix components and ADSC viability, differentiation and growth factor gene expression were evaluated. RESULTS: The effects of ADSCs on tenocytes were observed more in the microwound healing model, in which the rate of microwound healing and the expression of decorin, tenascin and collagens were significantly increased. The influence of tenocytes on ADSCs was also found in standard culture conditions: ADSCs were directed toward a tenogenic lineage, and growth factor expression increased. CONCLUSIONS: This study clarifies some aspects of the mutual interaction of ADSCs and tenocytes and provides in vitro evidence for a possible future application of ADSCs as a therapeutic strategy for tendon repair.


Asunto(s)
Tejido Adiposo/citología , Células Madre Mesenquimatosas/citología , Traumatismos de los Tendones/terapia , Tendones/fisiología , Cicatrización de Heridas/fisiología , Animales , Comunicación Celular , Diferenciación Celular , Células Cultivadas , Técnicas de Cocultivo , Colágeno/metabolismo , Decorina/metabolismo , Matriz Extracelular , Femenino , Ratas , Ratas Sprague-Dawley , Tenascina/metabolismo , Tendones/citología
11.
J Neurooncol ; 121(1): 119-27, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25261924

RESUMEN

Medulloblastomas (MBs) associated with the Wnt activation represent a subgroup with a favorable prognosis, but it remains unclear whether Wnt activation confers a less aggressive phenotype and/or enhances radiosensitivity. To investigate this issue, we evaluated the biological behavior of an MB cell line, UW228-1, stably transfected with human ß-catenin cDNA encoding a nondegradable form of ß-catenin (UW-B) in standard culture conditions and after radiation treatment. We evaluated the expression, transcriptional activity, and localization of ß-catenin in the stably transfected cells using immunofluorescence and WB. We performed morphological analysis using light and electron microscopy. We then analyzed changes in the invasiveness, growth, and mortality in standard culture conditions and after radiation. We demonstrated that (A) Wnt activation inhibited 97 % of the invasion capability of the cells, (B) the growth of the UW-B cells was statistically significantly lower than that of all the other control cells (p < 0.01), (C) the mortality of irradiated UW-B cells was statistically significantly higher than that of the controls and their nonirradiated counterparts (p < 0.05), and (D) morphological features of neuronal differentiation were observed in the Wnt-activated cells. In tissue samples, the Ki-67 labeling index (LI) was lower in ß-catenin-positive samples compared to non-ß-catenin positive ones. The Ki-67 LI median (LI = 40) of the nuclear ß-catenin-positive tumor samples was lower than that of non-nuclear ß-catenin-positive samples (LI = 50), but the difference was not statistically significant. Overall, our data suggest that activation of the Wnt pathway reduces the proliferation and invasion of MBs and increases the tumor's radiosensitivity.


Asunto(s)
Proliferación Celular/fisiología , Meduloblastoma/fisiopatología , Meduloblastoma/radioterapia , Tolerancia a Radiación/fisiología , Proteínas Wnt/metabolismo , Adolescente , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Niño , Preescolar , Técnica del Anticuerpo Fluorescente , Humanos , Lactante , Recién Nacido , Antígeno Ki-67/metabolismo , Meduloblastoma/patología , Microscopía Electrónica de Transmisión , Invasividad Neoplásica/fisiopatología , Neurogénesis/fisiología , Transfección , beta Catenina/genética , beta Catenina/metabolismo
12.
PLoS One ; 19(5): e0303035, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38820355

RESUMEN

Fracture non-unions affect many patients worldwide, however, known risk factors alone do not predict individual risk. The identification of novel biomarkers is crucial for early diagnosis and timely patient treatment. This study focused on the identification of microRNA (miRNA) related to the process of fracture healing. Serum of fracture patients and healthy volunteers was screened by RNA sequencing to identify differentially expressed miRNA at various times after injury. The results were correlated to miRNA in the conditioned medium of human bone marrow mesenchymal stromal cells (BMSCs) during in vitro osteogenic differentiation. hsa-miR-1246, hsa-miR-335-5p, and miR-193a-5p were identified both in vitro and in fracture patients and their functional role in direct BMSC osteogenic differentiation was assessed. The results showed no influence of the downregulation of the three miRNAs during in vitro osteogenesis. However, miR-1246 may be involved in cell proliferation and recruitment of progenitor cells. Further studies should be performed to assess the role of these miRNA in other processes relevant to fracture healing.


Asunto(s)
Biomarcadores , Diferenciación Celular , MicroARN Circulante , Células Madre Mesenquimatosas , MicroARNs , Osteogénesis , Humanos , Osteogénesis/genética , MicroARNs/sangre , MicroARNs/genética , Células Madre Mesenquimatosas/metabolismo , Biomarcadores/sangre , Masculino , MicroARN Circulante/sangre , MicroARN Circulante/genética , Femenino , Curación de Fractura/genética , Adulto , Fracturas Óseas/sangre , Fracturas Óseas/genética , Persona de Mediana Edad , Células Cultivadas , Proliferación Celular
13.
Blood Purif ; 35(1-3): 187-95, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23485859

RESUMEN

BACKGROUND: The effects of vitamin D receptor (VDR) and osteocalcin (OC) expression as well as VDR agonist (VDRA) therapy on circulating endothelial progenitor cells (EPCs) has not been elucidated yet. METHODS: We therefore analyzed EPCs in 30 healthy controls and 82 patients undergoing dialysis (no VDRA therapy: 28; oral calcitriol: 30, and intravenous paricalcitol, PCTA: 24). The percentage of EPCs (CD34+/CD133-/KDR+/CD45-) expressing VDR or OC, and VDR and OC expression defined by mean fluorescence intensity (MFI) were analyzed using flow cytometry. The in vitro effect of VDRAs was evaluated in EPCs isolated from each patient group. RESULTS: The percentage of VDR+ EPCs correlated positively with VDRA therapy and 25(OH)D, and negatively with diabetes, C-reactive protein, hemoglobin and osteopontin. VDR-MFI correlated positively with VDRA therapy, parathyroid hormone (PTH) and 25(OH)D, and negatively with diabetes and osteopontin. The percentage of OC+ EPCs correlated positively with the calcium score, PTH and phosphate, and negatively with 25(OH)D. OC-MFI correlated positively with calcium score, PTH, phosphate and hemoglobin, and negatively with albumin, 25(OH)D and osteopontin. Cell cultures from patients without VDRA therapy had the highest levels of calcium deposition and OC expression, which both significantly decreased following in vitro VDRA administration: in particular extracellular calcium deposition was only reduced by adding PCTA. CONCLUSIONS: Our data suggest that 25(OH)D serum levels and VDRA therapy influence VDR and OC expression on circulating EPCs. Since OC expression may contribute to vascular calcification, we hypothesize a putative protective role of VDRA therapy.


Asunto(s)
Células Endoteliales/efectos de los fármacos , Complejo Mediador/farmacología , Osteocalcina/genética , Receptores de Calcitriol/genética , Diálisis Renal , Insuficiencia Renal Crónica/tratamiento farmacológico , Células Madre/efectos de los fármacos , 25-Hidroxivitamina D 2/sangre , Antígenos CD/sangre , Antígenos CD/genética , Proteína C-Reactiva , Calcio/sangre , Estudios de Casos y Controles , Células Endoteliales/metabolismo , Células Endoteliales/patología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Osteocalcina/sangre , Osteopontina/sangre , Osteopontina/genética , Hormona Paratiroidea/sangre , Hormona Paratiroidea/genética , Receptores de Calcitriol/sangre , Insuficiencia Renal Crónica/sangre , Células Madre/metabolismo , Células Madre/patología
14.
Methods Mol Biol ; 2598: 1-7, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36355280

RESUMEN

Once damaged, cartilage has limited healing capability. This has led to a huge body of research that aims to repair or regenerate this important tissue. Despite the progress made, significant hurdles still need to be overcome. This chapter highlights some of the progress made, while elaborating on areas that need further research. The concept of translation and the route to clinical translation must be kept in mind if some of the promising preclinical research is to make it to routine clinical application.


Asunto(s)
Cartílago Articular , Ingeniería de Tejidos , Medicina Regenerativa , Andamios del Tejido
15.
Methods Mol Biol ; 2598: 65-73, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36355285

RESUMEN

Bone marrow-derived mesenchymal stromal cells (BM-MSC) are widely studied in the field of cartilage regeneration due to their capacity to differentiate into chondrocytes under specific in vitro culture conditions. This chapter describes the isolation of MSC from bone marrow aspirate, their expansion in monolayer, and the chondrogenic differentiation in pellet culture.


Asunto(s)
Médula Ósea , Células Madre Mesenquimatosas , Humanos , Células de la Médula Ósea , Condrogénesis , Diferenciación Celular , Condrocitos , Células Cultivadas
16.
Micromachines (Basel) ; 14(7)2023 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-37512632

RESUMEN

Since its discovery in 1989, RNA interference (RNAi) has become a widely used tool for the in vitro downregulation of specific gene expression in molecular biological research. This basically involves a complementary RNA that binds a target sequence to affect its transcription or translation process. Currently, various small RNAs, such as small interfering RNA (siRNA), micro RNA (miRNA), small hairpin RNA (shRNA), and PIWI interacting RNA (piRNA), are available for application on in vitro cell culture, to regulate the cells' gene expression by mimicking the endogenous RNAi-machinery. In addition, several biochemical, physical, and viral methods have been established to deliver these RNAs into the cell or nucleus. Since each RNA and each delivery method entail different off-target effects, limitations, and compatibilities, it is crucial to understand their basic mode of action. This review is intended to provide an overview of different nucleic acids and delivery methods for planning, interpreting, and troubleshooting of RNAi experiments.

17.
J Mol Graph Model ; 125: 108587, 2023 12.
Artículo en Inglés | MEDLINE | ID: mdl-37579519

RESUMEN

The glucocorticoid receptor (GR) is a nuclear receptor that controls critical biological processes by regulating the transcription of specific genes. GR transcriptional activity is modulated by a series of ligands and coenzymes, where a ligand can act as an agonist or antagonist. GR agonists, such as the glucocorticoids dexamethasone (DEX) and prednisolone, are widely prescribed to patients with inflammatory and autoimmune diseases. DEX is also used to induce osteogenic differentiation in vitro. Recently, it has been highlighted that DEX induces changes in the osteogenic differentiation of human mesenchymal stromal cells by downregulating the transcription factor SRY-box transcription factor 9 (SOX9) and upregulating the peroxisome proliferator-activated receptor γ (PPARG). SOX9 is fundamental in the control of chondrogenesis, but also in osteogenesis by acting as a dominant-negative of RUNX2. Many processes remain to be clarified during cell fate determination, such as the interplay between the key transcription factors. The main objective pursued by this work is to shed light on the interaction between GR and SOX9 in the presence and absence of DEX at an atomic level of resolution using molecular dynamics simulations. The outcome of this research could help the understanding of possible molecular interactions between GR and SOX9 and their role in the determination of cell fate. The results highlight the key residues at the interface between GR and SOX9 involved in the complexation process and shed light on the mechanism through which DEX modulates GR-SOX9 binding and exerts its biological activity.


Asunto(s)
Dexametasona , Receptores de Glucocorticoides , Humanos , Receptores de Glucocorticoides/genética , Dexametasona/farmacología , Simulación de Dinámica Molecular , Osteogénesis/genética , Factores de Transcripción/metabolismo , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo
18.
Clin Transl Med ; 13(1): e1161, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36629031

RESUMEN

BACKGROUND: Approximately 10% of all bone fractures result in delayed fracture healing or non-union; thus, the identification of biomarkers and prognostic factors is of great clinical interest. MicroRNAs (miRNAs) are known to be involved in the regulation of the bone healing process and may serve as functional markers for fracture healing. AIMS AND METHODS: This systematic review aimed to identify common miRNAs involved in fracture healing or non-union fractures using a qualitative approach. A systematic literature search was performed with the keywords 'miRNA and fracture healing' and 'miRNA and non-union fracture'. Any original article investigating miRNAs in fracture healing or non-union fractures was screened. Eventually, 82 studies were included in the qualitative analysis for 'miRNA and fracture healing', while 19 were selected for the 'miRNA and fracture non-union' category. RESULTS AND CONCLUSIONS: Out of 151 miRNAs, miR-21, miR-140 and miR-214 were the most investigated miRNAs in fracture healing in general. miR-31-5p, miR-221 and miR-451-5p were identified to be regulated specifically in non-union fractures. Large heterogeneity was detected between studies investigating the role of miRNAs in fracture healing or non-union in terms of patient population, sample types and models used. Nonetheless, our approach identified some miRNAs with the potential to serve as biomarkers for non-union fractures, including miR-31-5p, miR-221 and miR-451-5p. We provide a discussion of involved pathways and suggest on alignment of future research in the field.


Asunto(s)
Fracturas Óseas , MicroARNs , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Pronóstico , Curación de Fractura/genética , Fracturas Óseas/genética , Fracturas Óseas/terapia , Biomarcadores
19.
Tissue Eng Part B Rev ; 28(5): 949-965, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-34579558

RESUMEN

Musculoskeletal disorders are the most common reason of chronic pain and disability, representing an enormous socioeconomic burden worldwide. In this review, new biomedical application fields for Raman spectroscopy (RS) technique related to skeletal tissues are discussed, showing that it can provide a comprehensive profile of tissue composition in situ, in a rapid, label-free, and nondestructive manner. RS can be used as a tool to study tissue alterations associated to aging, pathologies, and disease treatments. The main advantage with respect to currently applied methods in clinics is its ability to provide specific information on molecular composition, which goes beyond other diagnostic tools. Being compatible with water, RS can be performed without pretreatment on unfixed, hydrated tissue samples, without any labeling and chemical fixation used in histochemical methods. This review first provides the description of the basic principles of RS as a biotechnology tool and is introduced into the field of currently available RS-based techniques, developed to enhance Raman signals. The main spectral processing, statistical tools, fingerprint identification, and available databases are mentioned. The recent literature has been analyzed for such applications of RS as tendon and ligaments, cartilage, bone, and tissue engineered constructs for regenerative medicine. Several cases of proof-of-concept preclinical studies have been described. Finally, advantages, limitations, future perspectives, and challenges for the translation of RS into clinical practice have been also discussed. Impact statement Raman spectroscopy (RS) is a powerful noninvasive tool giving access to molecular vibrations and characteristics of samples in a wavelength window of 600 to 3200 cm-1, thus giving access to a molecular fingerprint of biological samples in a nondestructive way. RS could not only be used in clinical diagnostics, but also be used for quality control of tissues and tissue-engineered constructs, reducing number of samples, time, and the variety of analysis required in the quality control chain before implantation.


Asunto(s)
Espectrometría Raman , Ingeniería de Tejidos , Humanos , Espectrometría Raman/métodos , Estudios Prospectivos , Cartílago , Agua
20.
Stem Cell Res Ther ; 13(1): 533, 2022 12 27.
Artículo en Inglés | MEDLINE | ID: mdl-36575539

RESUMEN

BACKGROUND: Bone marrow mesenchymal stromal cells (BMSCs) are promising for therapeutic use in cartilage repair, because of their capacity to differentiate into chondrocytes. Often, in vitro differentiation protocols employ the use of high amount of glucose, which does not reflect cartilage physiology. For this reason, we investigated how different concentrations of glucose can affect the chondrogenic differentiation of BMSCs in cell culture pellets. Additionally, we investigated how fructose could influence the chondrogenic differentiation in vitro. METHODS: BMSC were isolated from six donors and cultured in DMEM containing glucose at either 25 mM (HG), 5.5 mM (LG) or 1 mM (LLG), and 1% non-essential amino acids, 1% ITS+, in the presence of 100 nM dexamethasone, 50 µg/ml ascorbic acid-2 phosphate and 10 ng/ml TGF-ß1. To investigate the effect of different metabolic substrates, other groups were exposed to additional 25 mM fructose. The media were replaced every second day until day 21 when all the pellets were harvested for further analyses. Biochemical analysis for glycosaminoglycans into pellets and released in medium was performed using the DMMB method. Expression of GLUT3 and GLUT5 was assayed by qPCR and validated using FACS analysis and immunofluorescence in monolayer cultures. Chondrogenic differentiation was further confirmed by qPCR analysis of COL2A1, COL1A1, COL10A1, ACAN, RUNX2, SOX9, SP7, MMP13, and PPARG, normalized on RPLP0. Type 2 collagen expression was subsequently validated by immunofluorescence analysis. RESULTS: We show for the first time the presence of fructose transporter GLUT5 in BMSC and its regulation during chondrogenic commitment. Additionally, decreasing glucose concentration during chondrogenesis dramatically decreased the yield of differentiation. However, the use of fructose alone or together with low glucose concentrations does not limit cell differentiation, but on the contrary it might help in maintaining a stable chondrogenic phenotype comparable with the standard culture conditions (high glucose). CONCLUSION: This study provides evidence that BMSC express GLUT5 and differentially regulate GLUT3 in the presence of glucose variation. This study gives a better comprehension of BMSCs sugar use during chondrogenesis.


Asunto(s)
Médula Ósea , Células Madre Mesenquimatosas , Humanos , Transportador de Glucosa de Tipo 3/metabolismo , Condrogénesis , Glucosa/farmacología , Glucosa/metabolismo , Fructosa/farmacología , Fructosa/metabolismo , Condrocitos/metabolismo , Diferenciación Celular , Células Madre Mesenquimatosas/metabolismo , Células Cultivadas , Células de la Médula Ósea
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