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1.
ACS Nano ; 13(4): 3830-3838, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-30895772

RESUMO

Periodontitis is a common chronic inflammatory disease that affects tooth-supporting tissues. We engineer a multifunctional periodontal membrane for the guided tissue regeneration of lost periodontal tissues. The major drawback of current periodontal membranes is the lack of tissue regeneration properties. Here, a series of nanofibrous membranes based on poly(ε-caprolactone) with tunable biochemical and biophysical properties were developed for periodontal tissue regeneration. The engineered membranes were surface coated using biomimetic polydopamine to promote the adhesion of therapeutic proteins and cells. We demonstrate successful cellular localization on the surface of the engineered membrane by morphological patterning. Polydopamine accelerates osteogenic differentiation of dental-derived stem cells by promoting hydroxyapatite mineralization. Such multiscale designs can mimic the complex extracellular environment of periodontal tissue and serve as functional tissue constructs for periodontal regeneration. In a periodontal defect model in rats, our engineered periodontal membrane successfully promoted the regeneration of periodontal tissue and bone repair. Altogether, our data demonstrate that our biomimetic membranes have potential as protein/cell delivery platforms for periodontal tissue engineering.

2.
Adv Healthc Mater ; 6(24)2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29076281

RESUMO

Repair or regeneration of damaged nerves is still a challenging clinical task in reconstructive surgeries and regenerative medicine. Here, it is demonstrated that periodontal ligament stem cells (PDLSCs) and gingival mesenchymal stem cells (GMSCs) isolated from adult human periodontal and gingival tissues assume neuronal phenotype in vitro and in vivo via a subcutaneous transplantation model in nude mice. PDLSCs and GMSCs are encapsulated in a 3D scaffold based on alginate and hyaluronic acid hydrogels capable of sustained release of human nerve growth factor (NGF). The elasticity of the hydrogels affects the proliferation and differentiation of encapsulated MSCs within scaffolds. Moreover, it is observed that PDLSCs and GMSCs are stained positive for ßIII-tubulin, while exhibiting high levels of gene expression related to neurogenic differentiation (ßIII-tubulin and glial fibrillary acidic protein) via quantitative polymerase chain reaction (qPCR). Western blot analysis shows the importance of elasticity of the matrix and the presence of NGF in the neurogenic differentiation of encapsulated MSCs. In vivo, immunofluorescence staining for neurogenic specific protein markers confirms islands of dense positively stained structures inside transplanted hydrogels. As far as it is known, this study is the first demonstration of the application of PDLSCs and GMSCs as promising cell therapy candidates for nerve regeneration.


Assuntos
Alginatos/química , Gengiva/citologia , Ácido Hialurônico/química , Células-Tronco Mesenquimais/citologia , Regeneração Nervosa , Ligamento Periodontal/citologia , Tecidos Suporte , Animais , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Humanos , Hidrogéis/química , Masculino , Transplante de Células-Tronco Mesenquimais , Camundongos , Camundongos Nus , Modelos Animais , Adulto Jovem
3.
J Biomed Mater Res A ; 105(11): 2957-2967, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28639378

RESUMO

Mesenchymal stem cells (MSCs) derived from dental and orofacial tissues provide an alternative therapeutic option for craniofacial bone tissue regeneration. However, there is still a need to improve stem cell delivery vehicles to regulate the fate of the encapsulated MSCs for high quality tissue regeneration. Matrix elasticity plays a vital role in MSC fate determination. Here, we have prepared various hydrogel formulations based on alginate and gelatin methacryloyl (GelMA) and have encapsulated gingival mesenchymal stem cells (GMSCs) and human bone marrow MSCs (hBMMSCs) within these fabricated hydrogels. We demonstrate that addition of the GelMA to alginate hydrogel reduces the elasticity of the hydrogel mixture. While presence of GelMA in an alginate-based scaffold significantly increased the viability of encapsulated MSCs, increasing the concentration of GelMA downregulated the osteogenic differentiation of encapsulated MSCs in vitro due to decrease in the stiffness of the hydrogel matrix. The osteogenic suppression was rescued by addition of a potent osteogenic growth factor such as rh-BMP-2. In contrast, MSCs encapsulated in alginate hydrogel without GelMA were successfully osteo-differentiated without the aid of additional growth factors, as confirmed by expression of osteogenic markers (Runx2 and OCN), as well as positive staining using Xylenol orange. Interestingly, after two weeks of osteo-differentiation, hBMMSCs and GMSCs encapsulated in alginate/GelMA hydrogels still expressed CD146, an MSC surface marker, while MSCs encapsulated in alginate hydrogel failed to express any positive staining. Altogether, our findings suggest that it is possible to control the fate of encapsulated MSCs within hydrogels by tuning the mechanical properties of the matrix. We also reconfirmed the important role of the presence of inductive signals in guiding MSC differentiation. These findings may enable the design of new multifunctional scaffolds for spatial and temporal control over the fate and function of stem cells even post-transplantation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2957-2967, 2017.


Assuntos
Alginatos/química , Gelatina/química , Gengiva/citologia , Hidrogéis/química , Células-Tronco Mesenquimais/citologia , Tecidos Suporte/química , Materiais Biocompatíveis/química , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Células Imobilizadas/citologia , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Humanos , Masculino , Osteogênese , Engenharia Tecidual/métodos
4.
Development ; 142(21): 3734-45, 2015 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-26395480

RESUMO

Disrupted ERK1/2 signaling is associated with several developmental syndromes in humans. To understand the function of ERK2 (MAPK1) in the postmigratory neural crest populating the craniofacial region, we studied two mouse models: Wnt1-Cre;Erk2(fl/fl) and Osr2-Cre;Erk2(fl/fl). Wnt1-Cre;Erk2(fl/fl) mice exhibited cleft palate, malformed tongue, micrognathia and mandibular asymmetry. Cleft palate in these mice was associated with delay/failure of palatal shelf elevation caused by tongue malposition and micrognathia. Osr2-Cre;Erk2(fl/fl) mice, in which the Erk2 deletion is restricted to the palatal mesenchyme, did not display cleft palate, suggesting that palatal clefting in Wnt1-Cre;Erk2(fl/fl) mice is a secondary defect. Tongues in Wnt1-Cre;Erk2(fl/fl) mice exhibited microglossia, malposition, disruption of the muscle patterning and compromised tendon development. The tongue phenotype was extensively rescued after culture in isolation, indicating that it might also be a secondary defect. The primary malformations in Wnt1-Cre;Erk2(fl/fl) mice, namely micrognathia and mandibular asymmetry, are linked to an early osteogenic differentiation defect. Collectively, our study demonstrates that mutation of Erk2 in neural crest derivatives phenocopies the human Pierre Robin sequence and highlights the interconnection of palate, tongue and mandible development. Because the ERK pathway serves as a crucial point of convergence for multiple signaling pathways, our study will facilitate a better understanding of the molecular regulatory mechanisms of craniofacial development.


Assuntos
Sistema de Sinalização das MAP Quinases , Crista Neural/metabolismo , Síndrome de Pierre Robin/embriologia , Síndrome de Pierre Robin/metabolismo , Animais , Fissura Palatina/patologia , Feminino , Masculino , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Crista Neural/patologia , Síndrome de Pierre Robin/patologia , Língua/anormalidades
5.
PLoS One ; 9(4): e94607, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24736728

RESUMO

The aims of the study were to establish the prevalence of high bone mass (HBM) in a cohort of Spanish postmenopausal women (BARCOS) and to assess the contribution of LRP5 and DKK1 mutations and of common bone mineral density (BMD) variants to a HBM phenotype. Furthermore, we describe the expression of several osteoblast-specific and Wnt-pathway genes in primary osteoblasts from two HBM cases. A 0.6% of individuals (10/1600) displayed Z-scores in the HBM range (sum Z-score >4). While no mutation in the relevant exons of LRP5 was detected, a rare missense change in DKK1 was found (p.Y74F), which cosegregated with the phenotype in a small pedigree. Fifty-five BMD SNPs from Estrada et al. [NatGenet 44:491-501,2012] were genotyped in the HBM cases to obtain risk scores for each individual. In this small group of samples, Z-scores were found inversely related to risk scores, suggestive of a polygenic etiology. There was a single exception, which may be explained by a rare penetrant genetic variant, counterbalancing the additive effect of the risk alleles. The expression analysis in primary osteoblasts from two HBM cases and five controls suggested that IL6R, DLX3, TWIST1 and PPARG are negatively related to Z-score. One HBM case presented with high levels of RUNX2, while the other displayed very low SOX6. In conclusion, we provide evidence of lack of LRP5 mutations and of a putative HBM-causing mutation in DKK1. Additionally, we present SNP genotyping and expression results that suggest additive effects of several genes for HBM.


Assuntos
Densidade Óssea/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Pós-Menopausa/genética , Pós-Menopausa/fisiologia , Idoso , Alelos , Sequência de Aminoácidos , Animais , Desenvolvimento Ósseo/genética , Estudos de Coortes , Feminino , Regulação da Expressão Gênica , Loci Gênicos/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Osteoblastos/metabolismo , Fenótipo , Polimorfismo de Nucleotídeo Único , Espanha , Via de Sinalização Wnt/genética
7.
J Bone Miner Res ; 27(4): 950-3, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22190259

RESUMO

Fragility fractures resulting from low-trauma events such as a fall from standing height are associated with osteoporosis and are very common in older people, especially women. Three single nucleotide polymorphisms (SNPs) at the COL1A1 gene (rs1107946, rs11327935, and rs1800012) have been widely studied and previously associated with bone mineral density (BMD) and fracture. A rare haplotype (T-delT-T) of these three SNPs was found to be greatly overrepresented in fractured individuals compared with nonfractured controls, thus becoming a good candidate for predicting increased fracture risk. The aim of our study was to assess the association of this haplotype with fracture risk in Spanish individuals. We recruited two independent groups of ∼100 patients with hip fracture (a total of 203 individuals) and compared the genotype and haplotype distributions of the three SNPs in the fractured patients with those of 397 control individuals from the BARCOS Spanish cohort. We found no association with risk of fracture at the genotype level for any of the SNPs, and no differences in the SNP frequencies between the two groups. At the haplotype level, we found no association between the T-delT-T haplotype and fracture. However, we observed a small but significant (p = 0.03) association with another rare haplotype, G-insT-T, which was slightly overrepresented in the patient group.


Assuntos
Colágeno Tipo I/genética , Haplótipos/genética , Fraturas do Quadril/genética , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Masculino , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco
8.
J Bone Miner Res ; 26(5): 1133-44, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21542013

RESUMO

LRP5 is an osteoporosis susceptibility gene. Association analyses reveal that individual single-nucleotide polymorphisms (SNPs) determine variation in bone mineral density (BMD) among individuals as well as fracture risk. In a previous study, we identified a lumbar spine BMD-associated SNP, rs312009, located in the LRP5 5' region. A RUNX2 binding site was identified in this region by gel-shift experiments. Here we test the functionality of this SNP and examine whether RUNX2 is indeed a regulator of LRP5 expression. Gene reporter assays were used to test rs312009 functionality. Bioinformatic predictive tools and gel-shift and gene reporter assays were used to identify and characterize additional RUNX2 binding elements in the 3.3-kb region upstream of LRP5. Allelic differences in the transcriptional activity of rs312009 were observed in two osteoblastic cell lines, the T allele being a better transcriber than the C allele. RUNX2 cotransfection in HeLa cells revealed that the LRP5 5' region responded to RUNX2 in a dose-dependent manner and that the previously identified RUNX2 binding site participated in this response. Also, RUNX2 inhibition by RNAi led to nearly 60% reduction of endogenous LRP5 mRNA in U-2 OS cells. Four other RUNX2 binding sites were identified in the 5' region of LRP5. Luciferase experiments revealed the involvement of each of them in the RUNX2 response. The allelic differences observed point to the involvement of rs312009 as a functional SNP in the observed association. To our knowledge, this is the first time that the direct action of RUNX2 on LRP5 has been described. This adds evidence to previously described links between two important bone-regulating systems: the RUNX2 transcription-factor cascade and the Wnt signaling pathway.


Assuntos
Densidade Óssea/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica , Proteínas Relacionadas a Receptor de LDL/genética , Polimorfismo de Nucleotídeo Único/genética , Transcrição Genética , Pareamento de Bases/genética , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Humanos , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Dados de Sequência Molecular , Mutação/genética , Osteoblastos/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta/genética , Transfecção
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