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1.
J Clin Pediatr Dent ; 44(5): 364-372, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-33181847

RESUMO

OBJECTIVES: Temporomandibular disorder (TMD) is considered a functional disorder with multifactorial aspects. The goal of this study was to investigate if genetic polymorphisms in the COL2A1 gene could be associated with TMD in adolescents. STUDY DESIGN: The case group (TMD-affected) included individuals diagnosed with any of the following TMD subgroups according to the RDC/TMD criteria: myofascial pain, disc displacements and arthralgia. Genomic DNA for molecular analysis was extracted from buccal cells and genetic polymorphisms in COL2A1 were genotyped by real time polymerase chain reactions using the TaqMan assay. Data were analyzed using the Epi Info 3.5.7 and Stata software. RESULTS: 249 subjects were included in this study (148 subjects "affected" by TMD). There were no significant differences between the affected and unaffected individual (p>0.05), for TMD, arthralgia and myofascial pain however, rs2276454 was borderline in the genotype distribution (p=0.07) and was associated with disc displacement (p=0.03) in the allelic distribution. Recessive model showed significant differences between groups for with disc displacement (p=0.02). CONCLUSIONS: Genetic polymorphisms in COL2A1 are not associated with myofascial pain, arthralgia or TMD in adolescents but this study provides evidence that rs2276454 is involved in the disc displacement of the temporomandibular joint.


Assuntos
Luxações Articulares , Polimorfismo Genético , Transtornos da Articulação Temporomandibular , Síndrome da Disfunção da Articulação Temporomandibular , Adolescente , Artralgia , Colágeno Tipo II/genética , Dor Facial , Humanos , Mucosa Bucal , Articulação Temporomandibular , Transtornos da Articulação Temporomandibular/genética
2.
Acta Vet Scand ; 62(1): 49, 2020 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-32894162

RESUMO

BACKGROUND: Congenital bovine chondrodysplasia, also known as bulldog calf syndrome, is characterized by disproportionate growth of bones resulting in a shortened and compressed body, mainly due to reduced length of the spine and the long bones of the limbs. In addition, severe facial dysmorphisms including palatoschisis and shortening of the viscerocranium are present. Abnormalities in the gene collagen type II alpha 1 chain (COL2A1) have been associated with some cases of the bulldog calf syndrome. Until now, six pathogenic single-nucleotide variants have been found in COL2A1. Here we present a novel variant in COL2A1 of a Holstein calf and provide an overview of the phenotypic and allelic heterogeneity of the COL2A1-related bulldog calf syndrome in cattle. CASE PRESENTATION: The calf was aborted at gestation day 264 and showed generalized disproportionate dwarfism, with a shortened compressed body and limbs, and dysplasia of the viscerocranium; a phenotype resembling bulldog calf syndrome due to an abnormality in COL2A1. Whole-genome sequence (WGS) data was obtained and revealed a heterozygous 3513 base pair deletion encompassing 10 of the 54 coding exons of COL2A1. Polymerase chain reaction analysis and Sanger sequencing confirmed the breakpoints of the deletion and its absence in the genomes of both parents. CONCLUSIONS: The pathological and genetic findings were consistent with a case of "bulldog calf syndrome". The identified variant causing the syndrome was the result of a de novo mutation event that either occurred post-zygotically in the developing embryo or was inherited because of low-level mosaicism in one of the parents. The identified loss-of-function variant is pathogenic due to COL2A1 haploinsufficiency and represents the first structural variant causing bulldog calf syndrome in cattle. Furthermore, this case report highlights the utility of WGS-based precise diagnostics for understanding congenital disorders in cattle and the need for continued surveillance for genetic disorders in cattle.


Assuntos
Doenças dos Bovinos/genética , Colágeno Tipo II/genética , Deleção de Genes , Aborto Animal/patologia , Animais , Bovinos , Doenças dos Bovinos/congênito , Colágeno Tipo II/metabolismo
4.
PLoS One ; 15(6): e0234458, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32569264

RESUMO

We investigated the association of multiple single nucleotide polymorphisms (SNPs) with athlete status and power/speed performance in elite male youth soccer players (ESP) and control participants (CON) at different stages of maturity. ESP (n = 535; aged 8-23 years) and CON (n = 151; aged 9-26 years) were genotyped for 10 SNPs and grouped according to years from predicted peak-height-velocity (PHV), i.e. pre- or post-PHV, to determine maturity status. Participants performed bilateral vertical countermovement jumps, bilateral horizontal-forward countermovement jumps, 20m sprints and modified 505-agility tests. Compared to CON, pre-PHV ESP demonstrated a higher ACTN3 (rs1815739) XX ('endurance') genotype frequency distribution, while post-PHV ESP revealed a higher frequency distribution of the PPARA (rs4253778) C-allele, AGT (rs699) GG genotype and NOS3 (rs2070744) T-allele ('power' genotypes/alleles). BDNF (rs6265) CC, COL5A1 (rs12722) CC and NOS3 TT homozygotes sprinted quicker than A-allele carriers, CT heterozygotes and CC homozygotes, respectively. COL2A1 (rs2070739) CC and AMPD1 (rs17602729) GG homozygotes sprinted faster than their respective minor allele carrier counterparts in CON and pre-PHV ESP, respectively. BDNF CC homozygotes jumped further than T-allele carriers, while ESP COL5A1 CC homozygotes jumped higher than TT homozygotes. To conclude, we have shown for the first time that pre- and post-PHV ESP have distinct genetic profiles, with pre-PHV ESP more suited for endurance, and post-PHV ESP for power and speed (the latter phenotypes being crucial attributes for post-PHV ESP). We have also demonstrated that power, acceleration and sprint performance were associated with five SNPs, both individually and in combination, possibly by influencing muscle size and neuromuscular activation.


Assuntos
Atletas , Desempenho Atlético/fisiologia , Perfil Genético , Maturidade Sexual/fisiologia , Futebol , Aceleração , Actinina/genética , Adolescente , Estudos de Casos e Controles , Criança , Colágeno Tipo II/genética , Colágeno Tipo V/genética , Frequência do Gene , Humanos , Masculino , Força Muscular/genética , Polimorfismo de Nucleotídeo Único , Corrida/fisiologia , Adulto Jovem
5.
Neurochirurgie ; 66(3): 168-173, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32201238

RESUMO

PURPOSE: An accurate understanding of cellular biochemical changes in human intervertebral disc (IVD)s and the corresponding mechanisms during the developmental process still remain unknown and important for investigating the function of critical factors in normal IVD development as well as ascertaining the therapeutic targets for the IVD degeneration. METHODS: Under ethical conditions, human fetal cervical IVDs at 4, 5, and 6 months of pregnancy were collected at abortion surgery. Normal adult human C3-C7 cervical IVDs were taken from cadaveric donors. Sox9, Pax1, TGF-ß1 and type I/II collagen protein and RNA were detected. The number of positive cells was counted to calculate the optical density value for each factor. RESULTS: Sox9, Pax1, and TGF-ß1 expression in the IVD was remarkably reduced with the developmental stage. The location of high expression of Sox9, Pax1, and TGF-ß1 changed with the developmental stage, and migrated from the nucleus pulposus to the annulus fibrosus and endplate. Higher Sox9, Pax1, and TGF-ß1 expression was finally observed around the sclerotome of the vertebral body. The anabolism of type I/II collagens is significantly increased in the IVD in the mid-trimester fetus. CONCLUSIONS: Sox9, Pax1 and TGF-ß1 participate in the developmental process of the human IVD and vertebral body. However, these factors show a separate expression of mRNA and protein, suggesting that they are expressed in the strict time and spatial order.


Assuntos
Colágeno Tipo II/biossíntese , Colágeno Tipo I/biossíntese , Disco Intervertebral/crescimento & desenvolvimento , Disco Intervertebral/metabolismo , Fatores de Transcrição Box Pareados/biossíntese , Fatores de Transcrição SOX9/biossíntese , Fator de Crescimento Transformador beta1/biossíntese , Adulto , Cadáver , Colágeno Tipo I/genética , Colágeno Tipo II/genética , Feminino , Humanos , Imuno-Histoquímica , Disco Intervertebral/embriologia , Degeneração do Disco Intervertebral , Fatores de Transcrição Box Pareados/genética , Gravidez , Segundo Trimestre da Gravidez , RNA/biossíntese , RNA/genética , Fatores de Transcrição SOX9/genética , Fator de Crescimento Transformador beta1/genética
6.
Arthritis Rheumatol ; 72(7): 1123-1133, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32067417

RESUMO

OBJECTIVE: To investigate the effects of a young systemic environment and growth differentiation factor 11 (GDF-11) on aging cartilage. METHODS: A heterochronic parabiosis model (2-month-old mouse and 12-month-old mouse [Y/O]), an isochronic parabiosis model (12-month-old mouse and 12-month-old mouse [O/O]), and 12-month-old mice alone (O) were evaluated. Knee joints and chondrocytes from old mice were examined by radiography, histology, cell proliferation assays, immunohistochemistry, Western blotting, and quantitative reverse transcriptase-polymerase chain reaction 16 weeks after parabiosis surgery. GDF-11 was injected into 12-month-old mouse joints daily for 16 weeks. Cartilage degeneration, cell proliferation, and osteoarthritis-related gene expression were evaluated. RESULTS: Osteoarthritis Research Society International scores in old mice were significantly lower in the Y/O group than in the O/O and O groups (both P < 0.05). The percentage of 5-ethynyl-2'-deoxyuridine-positive chondrocytes in old mice was significantly higher in the Y/O group than in the other groups (P < 0.05). Type II collagen (CII) and SOX9 messenger RNA levels differed in cartilage from old mice in the Y/O group compared to the O/O and O groups (both P < 0.05). RUNX-2, CX, and matrix metalloproteinase 13 levels were significantly lower in cartilage from old mice in the Y/O group compared to the O/O and O groups (both P < 0.05). Similar results were obtained for protein expression levels and after GDF-11 treatment in vitro and in vivo. Phosphorylated Smad2/3 (pSmad2/3) levels were higher in the recombinant GDF-11-treated group than in the control group. CONCLUSION: A young systemic environment promotes chondrocyte proliferation and cartilage matrix synthesis in old mice. GDF-11, a "young factor," contributes to these effects through the up-regulation of pSmad2/3.


Assuntos
Envelhecimento/genética , Proteínas Morfogenéticas Ósseas/farmacologia , Cartilagem Articular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Fatores de Diferenciação de Crescimento/farmacologia , Osteoartrite do Joelho/genética , Parabiose , Adolescente , Idoso , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Artroplastia do Joelho , Proteínas Morfogenéticas Ósseas/metabolismo , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Colágeno Tipo II/efeitos dos fármacos , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Colágeno Tipo X/efeitos dos fármacos , Colágeno Tipo X/genética , Colágeno Tipo X/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Fatores de Diferenciação de Crescimento/metabolismo , Humanos , Técnicas In Vitro , Articulação do Joelho , Masculino , Metaloproteinase 13 da Matriz/efeitos dos fármacos , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Camundongos , Osteoartrite do Joelho/metabolismo , Fosforilação , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOX9/efeitos dos fármacos , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Proteína Smad2/efeitos dos fármacos , Proteína Smad2/metabolismo , Proteína Smad3/efeitos dos fármacos , Proteína Smad3/metabolismo , Joelho de Quadrúpedes , Adulto Jovem
7.
Exp Eye Res ; 191: 107907, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31899252

RESUMO

Rhegmatogenous retinal detachment (RRD) is the most common type of RD, the separation of neurosensory retina from the underlying retinal pigment epithelium. The RRD patients can be benefited from appropriate treatment if detected early, especially for the people predicted at high risk. In this study, we aimed to investigate the genetic association and clinical correlation of collagen type II alpha 1 (COL2A1) variants with sporadic RRD in a southern Chinese population. Totally 156 RRD patients and 254 control subjects were recruited, and 12 COL2A1 tag single nucleotide polymorphisms were genotyped by the TaqMan assay. The RRD patients had poorer visual acuity (P < 0.001) and lower intraocular pressure (IOP; P < 0.001) in their surgical eyes compared to the fellow eyes. The COL2A1 rs1793958 variant was significantly associated with RRD in the genotypic (P = 0.024), allelic (P = 0.011, odds ratio (OR) = 0.669), recessive (P = 0.011, OR = 0.384) and homozygous models (P = 0.007, OR = 0.348). RRD patients carrying the rs1793958 G allele had smaller retinal detachment area (P = 0.041) and smaller IOP differences (P = 0.046) between the surgical and fellow eyes compared to those carrying the wildtype AA genotype. In summary, this study revealed that the COL2A1 rs1793958 variant is associated with reduced risk of sporadic RRD, and patients carrying rs1793958 G allele have lower RRD severity.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Colágeno Tipo II/genética , Polimorfismo de Nucleotídeo Único , Descolamento Retiniano/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , China/epidemiologia , Feminino , Estudos de Associação Genética , Técnicas de Genotipagem , Humanos , Pressão Intraocular/fisiologia , Masculino , Pessoa de Meia-Idade , Descolamento Retiniano/diagnóstico por imagem , Estudos Retrospectivos , Ultrassonografia , Acuidade Visual/fisiologia
8.
Biosci Biotechnol Biochem ; 84(4): 695-702, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31809639

RESUMO

Emerging evidence has shown that microRNAs are important regulators in osteoarthritis (OA). Here, we investigated the function role of miR-455-3p in the pathogenesis of OA and the underlying molecular mechanisms. We first established the in vitro OA model using IL-1ß treated human chondrocyte cell line CHON-001. Using quantitative real time PCR, we observed the expression of miR-455-3p expression was up-regulated in the OA cartilage tissues and IL-1ß-treated chondrocytes. A series of function assays, including CCK-8 assay, flow cytometry, and ELISA assay showed that miR-455-3p contributed to IL-1ß-induced apoptosis and inflammation. Moreover, COL2A1 was confirmed as a target of miR-455-3p by luciferase reporter assay. Furthermore, COL2A1 knockdown reversed the effects of miR-455-3p inhibition, and aggravated the effects of miR-455-3p overexpression on IL-1ß-induced OA-like phenomenon. Taken together, these results revealed that miR-455-3p/COL2A1 axis might provide a novel molecular target for the treatment of OA.


Assuntos
Apoptose/genética , Condrócitos/citologia , Colágeno Tipo II/metabolismo , Inflamação/patologia , MicroRNAs/fisiologia , Osteoartrite/patologia , Regiões 3' não Traduzidas , Idoso , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Colágeno Tipo II/genética , Feminino , Técnicas de Silenciamento de Genes , Humanos , Técnicas In Vitro , Inflamação/metabolismo , Interleucina-1beta/farmacologia , Masculino , Pessoa de Meia-Idade , Osteoartrite/metabolismo
9.
Stem Cell Res Ther ; 10(1): 392, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31847882

RESUMO

BACKGROUND: Chondrogenesis represents a highly dynamic cellular process that leads to the establishment of various types of cartilage. However, when stress-related injuries occur, a rapid and efficient regeneration of the tissues is necessary to maintain cartilage integrity. Mesenchymal stem cells (MSCs) are known to exhibit high capacity for self-renewal and pluripotency effects, and thus play a pivotal role in the repair and regeneration of damaged cartilage. On the other hand, the influence of certain pathological conditions such as metabolic disorders on MSCs can seriously impair their regenerative properties and thus reduce their therapeutic potential. OBJECTIVES: In this investigation, we attempted to improve and potentiate the in vitro chondrogenic ability of adipose-derived mesenchymal stromal stem cells (ASCs) isolated from horses suffering from metabolic syndrome. METHODS: Cultured cells in chondrogenic-inductive medium supplemented with Cladophora glomerata methanolic extract were experimented for expression of the main genes and microRNAs involved in the differentiation process using RT-PCR, for their morphological changes through confocal and scanning electron microscopy and for their physiological homeostasis. RESULTS: The different added concentrations of C. glomerata extract to the basic chondrogenic inductive culture medium promoted the proliferation of equine metabolic syndrome ASCs (ASCsEMS) and resulted in chondrogenic phenotype differentiation and higher mRNA expression of collagen type II, aggrecan, cartilage oligomeric matrix protein, and Sox9 among others. The results reveal an obvious inhibitory effect of hypertrophy and a strong repression of miR-145-5p, miR-146-3p, and miR-34a and miR-449a largely involved in cartilage degradation. Treated cells additionally exhibited significant reduced apoptosis and oxidative stress, as well as promoted viability and mitochondrial potentiation. CONCLUSION: Chondrogenesis in EqASCsEMS was found to be prominent after chondrogenic induction in conditions containing C. glomerata extract, suggesting that the macroalgae could be considered for the enhancement of ASC cultures and their reparative properties.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Clorófitas/química , Condrogênese/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Síndrome Metabólica/patologia , Extratos Vegetais/farmacologia , Agrecanas/genética , Agrecanas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Clorófitas/metabolismo , Condrócitos/citologia , Condrócitos/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Cavalos , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Síndrome Metabólica/metabolismo , MicroRNAs/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/fisiologia , Extratos Vegetais/química , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo
10.
ACS Appl Mater Interfaces ; 11(49): 45479-45488, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31714732

RESUMO

Within the osteochondral interface, cellular and extracellular matrix gradients provide a biomechanical and biochemical niche for homeostatic tissue functions. Postnatal joint loading is critical for the development of such tissue gradients, leading to the formation of functional osteochondral tissues composed of superficial, middle, and deep zones of cartilage, and underlying subchondral bone, in a depth-dependent manner. In this regard, a novel, variable core-shell electrospinning strategy was employed to generate spatially controlled strain gradients within three-dimensional scaffolds under dynamic compressive loading, enabling the local strain-magnitude dependent, multiphenotypic stem cell differentiation. Human mesenchymal stem cells (hMSCs) were cultured in electrospun scaffolds with a linear or biphasic mechanical gradient, which was computationally engineered and experimentally validated. The cell/scaffold constructs were subjected to various magnitudes of dynamic compressive strains in a scaffold depth-dependent manner at a frequency of 1 Hz for 2 h daily for up to 42 days in osteogenic media. Spatially upregulated gene expression of chondrogenic markers (ACAN, COL2A1, PRG4) and glycosaminoglycan deposition was observed in the areas of greater compressive strains. In contrast, osteogenic markers (COL1A1, SPARC, RUNX2) and calcium deposition were downregulated in response to high local compressive strains. Dynamic mechanical analysis showed the maintenance of the engineered mechanical gradients only under dynamic culture conditions, confirming the potent role of biomechanical gradients in developing and maintaining a tissue gradient. These results demonstrate that multiphenotypic differentiation of hMSCs can be controlled by regulating local mechanical microenvironments, providing a novel strategy to recapitulate the gradient structure in osteochondral tissues for successful regeneration of damaged joints in vivo and facile development of interfacial tissue models in vitro.


Assuntos
Cartilagem/crescimento & desenvolvimento , Técnicas de Cultura de Células/métodos , Condrogênese/genética , Células-Tronco Mesenquimais/citologia , Agrecanas/genética , Diferenciação Celular/genética , Colágeno Tipo II/genética , Matriz Extracelular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Osteogênese/genética , Proteoglicanas/genética , Engenharia Tecidual/métodos , Tecidos Suporte
11.
PLoS One ; 14(10): e0223244, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31603905

RESUMO

The temporomandibular joint (TMJ) is a fibrocartilaginous tissue critical for chewing and speaking. In patients with temporomandibular disorders (TMDs), permanent tissue loss can occur. Recapitulating the complexity of TMDs in animal models is difficult, yet critical for the advent of new therapies. Synovial fluid from diseased human samples revealed elevated levels of tumor necrosis factor alpha (TNF-alpha). Here, we propose to recapitulate these findings in mice by subjecting murine TMJs with TNF-alpha or CFA (Complete Freund's Adjuvant) in mandibular condyle explant cultures and by local delivery in vivo using TMJ intra-articular injections. Both TNF-alpha and CFA delivery to whole mandibular explants and in vivo increased extracellular matrix deposition and increased cartilage thickness, while TNF-alpha treated explants had increased expression of inflammatory cytokines and degradative enzymes. Moreover, the application of TNF-alpha or CFA in both models reduced cell number. CFA delivery in vivo caused soft tissue inflammation, including pannus formation. Our work provides two methods of chemically induced TMJ inflammatory arthritis through a condyle explant model and intra-articular injection model that replicate findings seen in synovial fluid of human patients, which can be used for further studies delineating the mechanisms underlying TMJ pathology.


Assuntos
Artrite Experimental/imunologia , Cartilagem Articular/imunologia , Matriz Extracelular/imunologia , Transtornos da Articulação Temporomandibular/imunologia , Articulação Temporomandibular/imunologia , Proteína ADAMTS5/genética , Proteína ADAMTS5/imunologia , Adolescente , Adulto , Idoso , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/genética , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Colágeno Tipo II/genética , Colágeno Tipo II/imunologia , Colágeno Tipo X/genética , Colágeno Tipo X/imunologia , Modelos Animais de Doenças , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/patologia , Feminino , Adjuvante de Freund/administração & dosagem , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Humanos , Interleucinas/genética , Interleucinas/imunologia , Masculino , Côndilo Mandibular/efeitos dos fármacos , Côndilo Mandibular/imunologia , Côndilo Mandibular/patologia , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Líquido Sinovial/imunologia , Articulação Temporomandibular/efeitos dos fármacos , Articulação Temporomandibular/patologia , Transtornos da Articulação Temporomandibular/genética , Transtornos da Articulação Temporomandibular/patologia , Técnicas de Cultura de Tecidos , Fator de Necrose Tumoral alfa/administração & dosagem
12.
Cell Physiol Biochem ; 53(4): 623-637, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31550089

RESUMO

BACKGROUND/AIMS: In articular cartilage, chondrocytes are the predominant cell type. A long-term stay in space can lead to bone loss and cartilage breakdown. Due to the poor regenerative capacity of cartilage, this may impair the crewmembers' mobility and influence mission activities. Beside microgravity other factors such as cosmic radiation and vibration might be important for cartilage degeneration. Vibration at different frequencies showed various effects on cartilage in vivo, but knowledge about its impact on chondrocytes in vitro is sparse. METHODS: Human chondrocytes were exposed to a vibration device, simulating the vibration profile occurring during parabolic flights, for 24 h (VIB) and compared to static controls. Phase-contrast microscopy, immunofluorescence, F-actin and TUNEL staining as well as quantitative real-time PCR were performed to examine effects on morphology, cell viability and shape as well as gene expression. The results were compared to earlier studies using semantic analyses. RESULTS: No morphological changes or cytoskeletal alterations were observed in VIB and no apoptotic cells were found. A reorganization and increase in fibronectin were detected in VIB samples by immunofluorescence technique. PXN, VCL, ANXA1, ANXA2, BAX, and BCL2 revealed differential regulations. CONCLUSION: Long-term VIB did not damage human chondrocytes in vitro. The reduction of ANXA2, and up-regulation of ANXA1, PXN and VCL mRNAs suggest that long-term vibration might even positively influence cultured chondrocytes.


Assuntos
Condrócitos/metabolismo , Vibração , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Anexina A2/genética , Anexina A2/metabolismo , Linhagem Celular , Condrócitos/citologia , Condrócitos/patologia , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Redes Reguladoras de Genes , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Vimentina/genética , Vimentina/metabolismo
13.
Int J Mol Sci ; 20(15)2019 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-31382618

RESUMO

The temporomandibular joint (TMJ), which is biomechanically related to dental occlusion, is often insulted by osteoarthritis (OA). This study was conducted to clarify the relationship between Indian hedgehog (Ihh) and parathyroid hormone receptor 1 (PTH1R) signaling in modulating the enhanced chondrocyte terminal differentiation in dental stimulated TMJ osteoarthritic cartilage. A gain- and loss-of-function strategy was used in an in vitro model in which fluid flow shear stress (FFSS) was applied, and in an in vivo model in which the unilateral anterior cross-bite (UAC) stimulation was adopted. Ihh and PTH1R signaling was modulated through treating the isolated chondrocytes with inhibitor/activator and via deleting Smoothened (Smo) and/or Pth1r genes in mice with the promoter gene of type 2 collagen (Col2-CreER) in the tamoxifen-inducible pattern. We found that both FFSS and UAC stimulation promoted the deep zone chondrocytes to undergo terminal differentiation, while cells in the superficial zone were robust. We demonstrated that the terminal differentiation process in deep zone chondrocytes promoted by FFSS and UAC was mediated by the enhanced Ihh signaling and declined PTH1R expression. The FFSS-promoted terminal differentiation was suppressed by administration of the Ihh inhibitor or PTH1R activator. The UAC-promoted chondrocytes terminal differentiation and OA-like lesions were rescued in Smo knockout, but were enhanced in Pth1r knockout mice. Importantly, the relieving effect of Smo knockout mice was attenuated when Pth1r knockout was also applied. Our data suggest a chondrocyte protective effect of suppressing Ihh signaling in TMJ OA cartilage which is dependent on PTH1R signaling.


Assuntos
Proteínas Hedgehog/genética , Osteoartrite/genética , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Receptor Smoothened/genética , Animais , Cartilagem Articular/crescimento & desenvolvimento , Cartilagem Articular/metabolismo , Diferenciação Celular/genética , Condrócitos/metabolismo , Condrócitos/patologia , Condrogênese/genética , Colágeno Tipo II/genética , Oclusão Dentária , Humanos , Camundongos , Camundongos Knockout , Osteoartrite/patologia , Transdução de Sinais/genética , Estresse Mecânico , Articulação Temporomandibular/crescimento & desenvolvimento , Articulação Temporomandibular/metabolismo
14.
Hum Mutat ; 40(12): 2344-2352, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31389106

RESUMO

Campomelic dysplasia (CD) is an autosomal dominant, perinatal lethal skeletal dysplasia characterized by a small chest and short long bones with bowing of the lower extremities. CD is the result of heterozygosity for mutations in the gene encoding the chondrogenesis master regulator, SOX9. Loss-of-function mutations have been identified in most CD cases so it has been assumed that the disease results from haploinsufficiency for SOX9. Here, we identified distal truncating SOX9 mutations in four unrelated CD cases. The mutations all leave the dimerization and DNA-binding domains intact and cultured chondrocytes from three of the four cases synthesized truncated SOX9. Relative to CD resulting from haploinsufficiency, there was decreased transactivation activity toward a major transcriptional target, COL2A1, consistent with the mutations exerting a dominant-negative effect. For one of the cases, the phenotypic consequence was a very severe form of CD, with a pronounced effect on vertebral and limb development. The data identify a novel molecular mechanism of disease in CD in which the truncated protein leads to a distinct and more significant effect on SOX9 function.


Assuntos
Displasia Campomélica/genética , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Sequenciamento Completo do Exoma/métodos , Displasia Campomélica/metabolismo , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Colágeno Tipo II/genética , Feminino , Haploinsuficiência , Humanos , Gravidez , Diagnóstico Pré-Natal , Deleção de Sequência
15.
Curr Pharm Biotechnol ; 20(14): 1194-1202, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31400264

RESUMO

OBJECTIVE: Transfersomes are highly flexible vesicles that are capable of passing through pores smaller than their own sizes due to their elastochemical characteristics, and thus play a key role in drug delivery to the skin. METHODS: In this study, we used transdermal delivery of growth hormone-encapsulated transferosomes (F1 and F2) as antiaging strategy, with the resulting effects being subsequently evaluated. The size, distribution and zeta potential of the particles, together with the in vitro skin permeation and biological activity of the growth hormone loaded onto the transfersomes were measured. RESULTS: The data demonstrated that treatment of fibroblasts with encapsulated hGH increased cell migration, proliferation and collagen I and III gene expression. According to our results, the maximum amount of growth hormone that passes through the skin during a 24 h time period was 489.54 and 248.46 ng/cm3, for the F1 and F2 transfersomes, respectively. In addition, it was determined that F1 formula as the more efficient carrier, showed no toxicity against cells. With regard to fibroblasts, as one of the most important cells involved in collagen synthesis, skin aging and wound healing, concentrations of growth hormone encapsulated in transferosomes that had an effect on fibroblast growth and division, were determined. The results demonstrated that effective concentrations of the encapsulated growth hormone increased the expression of collagen I and collagen III genes. CONCLUSION: Furthermore, analyzing the rate of fibroblast cell migration showed that migration increased significantly at 700 ng/ml growth hormone concentrations, as compared to that of the control.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Fibroblastos/metabolismo , Hormônio do Crescimento Humano/administração & dosagem , Absorção Cutânea/efeitos dos fármacos , Administração Cutânea , Animais , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo II/genética , Liberação Controlada de Fármacos , Fibroblastos/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Hormônio do Crescimento Humano/farmacologia , Humanos , Lipossomos , Cultura Primária de Células , Ratos Sprague-Dawley , Envelhecimento da Pele/efeitos dos fármacos , Cicatrização/efeitos dos fármacos
16.
Matrix Biol ; 83: 77-96, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31381970

RESUMO

Cartilage remodelling and chondrocyte differentiation are tightly linked to angiogenesis during bone development and endochondral ossification. To investigate whether collagenase-mediated cleavage of the major cartilage collagen (collagen II) plays a role in this process, we generated a knockin mouse in which the mandatory collagenase cleavage site at PQG775↓776LAG, was mutated to PPG775↓776MPG (Col2a1Bailey). This approach blocked collagen II cleavage, and the production of putative collagen II matrikines derived from this site, without modifying matrix metalloproteinase expression or activity. We report here that this mouse (Bailey) is viable. It has a significantly expanded growth plate and exhibits delayed and abnormal angiogenic invasion into the growth plate. Deeper electron microscopy analyses revealed that, at around five weeks of age, a small number of blood vessel(s) penetrate into the growth plate, leading to its abrupt shrinking and the formation of a bony bridge. Our results from in vitro and ex vivo studies suggest that collagen II matrikines stimulate the normal branching of endothelial cells and promote blood vessel invasion at the chondro-osseous junction. The results further suggest that failed collagenolysis in Bailey leads to expansion of the hypertrophic zone and formation of a unique post-hypertrophic zone populated with chondrocytes that re-enter the cell cycle and proliferate. The biological rescue of this in vivo phenotype features the loss of a substantial portion of the growth plate through aberrant ossification, and narrowing of the remaining portion that leads to limb deformation. Together, these data suggest that collagen II matrikines stimulate angiogenesis in skeletal growth and development, revealing novel strategies for stimulating angiogenesis in other contexts such as fracture healing and surgical applications.


Assuntos
Condrócitos/citologia , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Colagenases/metabolismo , Lâmina de Crescimento/anormalidades , Animais , Diferenciação Celular , Proliferação de Células , Colágeno Tipo II/química , Feminino , Técnicas de Introdução de Genes , Lâmina de Crescimento/irrigação sanguínea , Masculino , Camundongos , Neovascularização Fisiológica , Osteogênese
17.
Macromol Biosci ; 19(8): e1900099, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31298816

RESUMO

Rational design and development of tailorable simple synthesis process remains a centerpiece of investigational efforts toward engineering advanced hydrogels. In this study, a green and scalable synthesis approach is developed to formulate a set of gelatin-based macroporous hybrid hydrogels. This approach consists of four sequential steps starting from liquid-phase pre-crosslinking/grafting, unidirectional freezing, freeze-drying, and finally post-curing process. The chemical crosslinking mainly involves between epoxy groups of functionalized polyethylene glycol and functional groups of gelatin both in liquid and solid state. Importantly, this approach allows to accommodate different polymers, chitosan or hydroxyethyl cellulose, under identical benign condition. Structural and mechanical anisotropy can be tuned by the selection of polymer constituents. Overall, all hydrogels show suitable structural stability, good swellability, high porosity and pore interconnectivity, and maintenance of mechanical integrity during 3-week-long hydrolytic degradation. Under compression, hydrogels exhibit robust mechanical properties with nonlinear elasticity and stress-relaxation behavior and show no sign of mechanical failure under repeated compression at 50% deformation. Biological experiment with human bone marrow mesenchymal stromal cells (hMSCs) reveals that hydrogels are biocompatible, and their physicomechanical properties are suitable to support cells growth, and osteogenic/chondrogenic differentiation, demonstrating their potential application for bone and cartilage regenerative medicine toward clinically relevant endpoints.


Assuntos
Materiais Biocompatíveis/síntese química , Condrogênese/efeitos dos fármacos , Gelatina/química , Hidrogéis/síntese química , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Anisotropia , Materiais Biocompatíveis/farmacologia , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Quitosana/química , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Condrogênese/genética , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Expressão Gênica , Humanos , Hidrogéis/farmacologia , Teste de Materiais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/genética , Polietilenoglicóis/química , Porosidade , Estresse Mecânico , Engenharia Tecidual , Tecidos Suporte
18.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(7): 694-696, 2019 Jul 10.
Artigo em Chinês | MEDLINE | ID: mdl-31302913

RESUMO

OBJECTIVE: To explore the molecular basis for a pedigree affected with spondyloepiphyseal dysplasia congenita (SEDC). METHODS: The proband was subjected to whole exome sequencing. Suspected variant was verified by Sanger sequencing. RESULTS: All patients from the pedigree were found to carry a novel missense variant c.1394G>C (p.Gly465Ala) of the COL2A1 gene. The variant was not reported previously. Provean, Polyphen-2 and Mutation Taster software predicted that the variant is highly likely to be pathogenic. CONCLUSION: The c.1394G>C (p.Gly465Ala) variant of the COL2A1 gene probably underlies the SEDC in this pedigree.


Assuntos
Colágeno Tipo II/genética , Osteocondrodisplasias/congênito , Grupo com Ancestrais do Continente Asiático , Humanos , Osteocondrodisplasias/genética , Linhagem
19.
Clin Rheumatol ; 38(10): 2897-2907, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31236747

RESUMO

INTRODUCTION/OBJECTIVES: Articular cartilage is the target tissue of osteoarthritis (OA), and because it lacks capillary networks, the microenvironment is hypoxic. Hypoxia inducible factor-1 alpha (HIF-1α) regulates the homeostasis of this tissue. The aim of this study was to investigate whether genetic polymorphisms of the HIF-1α signaling pathway are involved in the development of knee OA. METHOD: We performed a case-control association study and genotyped 134 knee OA patients and 267 healthy controls. All participants were genotyped in order to evaluate 42 SNPs from 22 genes involved in the HIF-1α signaling pathway using the OpenArray technology. Gene-gene interactions (epistasis) were analyzed using the multifactor dimensionality reduction (MDR) method. RESULTS: The MDR analysis showed epistasis between AKT2 (rs8100018) and IGF1 (rs2288377), AKT2 (rs8100018) and IGF1 (rs35767), IGF1 (rs35767) and COL2A1 (rs1793953), and between GSK3B (rs6438552) and IGF1 (rs35767) polymorphisms, with information gain values of 21.24%, 8.37%, 9.93%, and 5.73%, respectively. Additionally, our model allowed us to identify high- and low-risk genotypes among COL2A1 rs1793953, GSK3B rs6438552, AKT2 rs8100018, and IGF1 rs35767 polymorphisms. CONCLUSIONS: Knowing the interactions of these polymorphisms involved in HIF-1α signaling pathway could provide a new diagnostic support tool to identify individuals at high risk of developing knee OA.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Osteoartrite do Joelho/genética , Osteoartrite do Joelho/fisiopatologia , Polimorfismo de Nucleotídeo Único , Transdução de Sinais , Adulto , Capilares/patologia , Estudos de Casos e Controles , Colágeno Tipo II/genética , Epistasia Genética , Feminino , Genótipo , Glicogênio Sintase Quinase 3 beta/genética , Haplótipos , Homeostase , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fator de Crescimento Insulin-Like I/genética , Masculino , México , Pessoa de Meia-Idade , Modelos Genéticos , Proteínas Proto-Oncogênicas c-akt/genética , Risco
20.
Tissue Eng Regen Med ; 16(3): 285-299, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31205857

RESUMO

Background: This study aimed to observe the cartilaginous matrix production in SRY (sex determining region Y)-box 9 (SOX9)- and/or telomerase reverse transcriptase (TERT)-transfected chondrocytes from monolayer to three-dimensional (3D) culture. Methods: The genes were transferred into chondrocytes at passage-1 (P1) via lipofection. The post-transfected chondrocytes (SOX9-, TERT- and SOX9/TERT) were analysed at P1, P2 and P3. The non-transfected group was used as control. The 3D culture was established using the chondrocytes seeded in a disc-shaped PLGA/fibrin and PLGA scaffolds. The resulting 3D "cells-scaffolds" constructs were analysed at week-1, -2 and -3. The histoarchitecture was evaluated using haematoxylin and eosin, alcian blue and safranin o stains. The quantitative sulphated glycosaminoglycan (sGAG) content was measured using biochemical assay. The cartilage-specific markers expression were analysed via real-time polymerase chain reaction. Results: All monolayer cultured chondrocytes showed flattened, fibroblast-like appearance throughout passages. Proteoglycan and sGAG were not detected at the pericellular matrix region of the chondrocytes. The sGAG content assay indicated the matrix production depletion in the culture. The cartilage-specific markers, COL2A1 and ACAN, were downregulated. However, the dedifferentiation marker, COL1A1 was upregulated. In 3D "cells-scaffolds" constructs, regardless of transfection groups, chondrocytes seeded in PLGA/fibrin showed a more uniform distribution and produced denser matrix than the PLGA group especially at week-3. Both sGAG and proteoglycan were clearly visualised in the constructs, supported by the increment of sGAG content, quantitatively. Both COL2A1 and ACAN were upregulated in SOX9/TERT-PLGA and SOX9/TERT-PLGA/fibrin respectively. While, COL1A1 was downregulated in SOX9/TERT-PLGA. Conclusion: These findings indicated that the SOX9/TERT-transfected chondrocytes incorporation into 3D scaffolds facilitates the cartilage regeneration which is viable structurally and functionally.


Assuntos
Condrócitos/metabolismo , Fatores de Transcrição SOX9/genética , Telomerase/genética , Transfecção , Agrecanas/genética , Agrecanas/metabolismo , Animais , Células Cultivadas , Condrócitos/citologia , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Fibrina , Fibroblastos/citologia , Regulação da Expressão Gênica , Glicosaminoglicanos , Proteoglicanas , Coelhos , Ratos , Tecidos Suporte
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