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1.
Cell Mol Biol Lett ; 27(1): 46, 2022 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-35690719

RESUMO

The submandibular gland (SMG) and the sublingual gland (SLG) are two of the three major salivary glands in mammals. In mice, they are adjacent to each other and open into the oral cavity, producing saliva to lubricate the mouth and aid in food digestion. Though salivary gland dysfunction accompanied with fibrosis and metabolic disturbance is common in clinic, in-depth mechanistic research is lacking. Currently, research on how to rescue salivary function is challenging, as it must resort to using terminally differentiated acinar cells or precursor acinar cells with unknown differentiation. In this study, we established reversely immortalized mouse primary SMG cells (iSMGCs) and SLG cells (iSLGCs) on the first postnatal day (P0). The iSMGCs and iSLGCs grew well, exhibited many salivary gland characteristics, and retained the metabolism-related genes derived from the original tissue as demonstrated using transcriptome sequencing (RNA-seq) analysis. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of these two cell lines, which overlapped with those of the SMG and SLG, were enriched in cysteine and methionine metabolism. Furthermore, we investigated the role of bone morphogenetic protein 9 (BMP9), also known as growth differentiation factor 2(Gdf2), on metabolic and fibrotic functions in the SMG and SLG. We demonstrated that iSMGCs and iSLGCs presented promising adipogenic and fibrotic responses upon BMP9/Gdf2 stimulation. Thus, our findings indicate that iSMGCs and iSLGCs faithfully reproduce characteristics of SMG and SLG cells and present a promising prospect for use in future study of salivary gland metabolism and fibrosis upon BMP9/Gdf2 stimulation.


Assuntos
Fator 2 de Diferenciação de Crescimento , Glândula Sublingual , Animais , Linhagem Celular , Fibrose , Fator 2 de Diferenciação de Crescimento/metabolismo , Mamíferos , Camundongos , Glândulas Salivares/metabolismo , Glândula Sublingual/metabolismo
2.
J Cell Mol Med ; 25(5): 2666-2678, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33605035

RESUMO

Teeth arise from the tooth germ through sequential and reciprocal interactions between immature epithelium and mesenchyme during development. However, the detailed mechanism underlying tooth development from tooth germ mesenchymal cells (TGMCs) remains to be fully understood. Here, we investigate the role of Wnt/ß-catenin signalling in BMP9-induced osteogenic/odontogenic differentiation of TGMCs. We first established the reversibly immortalized TGMCs (iTGMCs) derived from young mouse mandibular molar tooth germs using a retroviral vector expressing SV40 T antigen flanked with the FRT sites. We demonstrated that BMP9 effectively induced expression of osteogenic markers alkaline phosphatase, collagen A1 and osteocalcin in iTGMCs, as well as in vitro matrix mineralization, which could be remarkably blunted by knocking down ß-catenin expression. In vivo implantation assay revealed that while BMP9-stimulated iTGMCs induced robust formation of ectopic bone, knocking down ß-catenin expression in iTGMCs remarkably diminished BMP9-initiated osteogenic/odontogenic differentiation potential of these cells. Taken together, these discoveries strongly demonstrate that reversibly immortalized iTGMCs retained osteogenic/odontogenic ability upon BMP9 stimulation, but this process required the participation of canonical Wnt signalling both in vitro and in vivo. Therefore, BMP9 has a potential to be applied as an efficacious bio-factor in osteo/odontogenic regeneration and tooth engineering. Furthermore, the iTGMCs may serve as an important resource for translational studies in tooth tissue engineering.


Assuntos
Fator 2 de Diferenciação de Crescimento/genética , Células-Tronco Mesenquimais/metabolismo , Odontogênese/genética , Osteogênese/genética , Germe de Dente/citologia , Via de Sinalização Wnt , Animais , Diferenciação Celular , Linhagem Celular , Transformação Celular Neoplásica , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Fator 2 de Diferenciação de Crescimento/metabolismo , Xenoenxertos , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos
3.
Int J Med Sci ; 16(4): 567-575, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31171908

RESUMO

Bone tissue engineering requires a combination of cells, efficient biochemical and physicochemical factors, and biocompatible scaffolds. In this study, we evaluated the potential use of injectable Matrigel as a scaffold for the delivery of rat dental follicle stem/precursor cells (rDFSCs) transduced by bone morphogenetic protein (BMP) 9 to enhance osteogenic differentiation in vitro and promote ectopic bone formation in vivo. Recombinant adenovirus was used to overexpress BMP9 in rDFSCs. Alkaline phosphatase activity was measured using a histochemical staining assay and a chemiluminescence assay kit. Quantitative real-time polymerase chain reaction was used to determine mRNA expression levels of bone-related genes including distal-less homeobox 5 (DLX5), osteopontin (OPN), osterix (Osx), and runt-related transcription factor 2 (Runx2). Matrix mineralization was examined by Alizarin Red S staining. rDFSCs proliferation was analyzed using the Cell Counting Kit-8 assay. Subcutaneous implantation of rDFSCs-containing Matrigel scaffolds was used, and micro-computed tomography analysis, histological evaluation, and trichrome staining of implants extracted at 6 weeks were performed. We found that BMP9 enhanced alkaline phosphatase activity and mineralization in rDFSCs. The expression of bone-related genes (DLX5, OPN, Osx, and Runx2) was also increased as a result of BMP9 stimulation. Micro-computed tomography analysis and histological evaluation revealed that the bone masses retrieved from BMP9-overexpressing rDFSCs were significantly more pronounced in those with than in those without Matrigel. Our results suggest that BMP9 effectively promote osteogenic differentiation of rDFSCs, and Matrigel facilitate BMP9-induced osteogenesis of rDFSCs in vivo.


Assuntos
Fator 2 de Diferenciação de Crescimento/genética , Osteogênese/efeitos dos fármacos , Transplante de Células-Tronco , Alicerces Teciduais , Animais , Diferenciação Celular/efeitos dos fármacos , Colágeno/farmacologia , Saco Dentário/citologia , Combinação de Medicamentos , Fator 2 de Diferenciação de Crescimento/farmacologia , Humanos , Laminina/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/genética , Proteoglicanas/farmacologia , Ratos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Engenharia Tecidual , Microtomografia por Raio-X
4.
J Oral Maxillofac Surg ; 77(3): 499-514, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30476490

RESUMO

PURPOSE: Insufficient bone volume compromises the success rate and osseointegration of immediate implantation. The objective of the present study was to engineer bone tissue by using adipose-derived stem cell (ASC) sheets and autologous platelet-rich fibrin (PRF) to enhance new bone formation and osseointegration around dental implants. MATERIAL AND METHODS: The proliferation and osteogenic potential of ASCs treated with autologous PRF were evaluated with CCK-8 assays, alkaline phosphatase staining, and real-time quantitative polymerase chain reaction. A 3-wall bone defect around each immediate implant was generated in the mandible and randomly treated with ASC sheets plus PRF (group A), ASC sheets only (group B), PRF only (group C), or no treatment (group D). Micro-computed tomography, biomechanical tests, fluorescent bone labeling, and histologic assessments were performed to evaluate bone regeneration capacity. RESULTS: The proliferation and osteogenic potential of canine ASCs were markedly enhanced by PRF. Group A exhibited considerably more new bone formation and re-osseointegration (41.17 ± 1.44 and 55.06 ± 0.06%, respectively) than did the other 3 groups. Fluorescent labeling showed that the most rapid bone remodeling activity occurred in group A (P < .05). CONCLUSION: These results suggest that sheets of ASC combined with autologous PRF could be a promising tissue-engineering strategy for bone formation in immediate implantation.


Assuntos
Implantes Dentários , Células-Tronco Mesenquimais , Regeneração Óssea , Osteogênese , Fibrina Rica em Plaquetas , Microtomografia por Raio-X
5.
Proteomics ; 18(3-4)2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29327447

RESUMO

Hypoxic preconditioning is commonly applied to enhance mesenchymal stem cells (MSCs) therapeutic effect before transplantation. Elucidating the effect of hypoxic preconditioning would be beneficial for improved application. However, the influence of hypoxia on dental tissue derived MSCs cultured in 3D was unknown. Thus, the present study is to investigate gene expression and proteome of dental pulp stem cells (DPSCs) after hypoxic preconditioning. DPSCs were isolated, cultured in a 3D system under the normoxic and hypoxic conditions. The gene expression was examined with reverse transcription polymerase chain reaction, and the proteome was analyzed using iTRAQ-based mass spectrometry. The expressions of HIF-1α, VEGFA, KDR at mRNA level was upregulated while BMP-2 was downregulated. Two thousand one hundred and fifteen proteins were identified and 57 proteins exhibited significant differences after hypoxic preconditioning (30 up-regulated, 27 down-regulated). Bioinformatic analysis revealed the majority of up-regulated proteins are involved in cellular process, angiogenesis, protein binding and transport, regulation of response to stimulus, metabolic processes, and immune response. Increased IL-6 and decreased TGF-1ß protein expression under hypoxic condition were verified by ELISA. Hypoxic preconditioning partly affected the gene and protein expression in DPSCs under 3D culture and may enhance the efficacy of MSCs transplantation.


Assuntos
Polpa Dentária/citologia , Espectrometria de Massas/métodos , Células-Tronco Mesenquimais/metabolismo , Proteoma/análise , Técnicas de Cultura de Células/métodos , Células Cultivadas , Polpa Dentária/metabolismo , Regulação da Expressão Gênica , Hipóxia , Marcação por Isótopo , Células-Tronco Mesenquimais/citologia , Oxigênio/metabolismo
6.
Adv Healthc Mater ; 12(30): e2301733, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37660274

RESUMO

Since the microgap between implant and surrounding connective tissue creates the pass for pathogen invasion, sustained pathological stimuli can accelerate macrophage-mediated inflammation, therefore affecting peri-implant tissue regeneration and aggravate peri-implantitis. As the transmucosal component of implant, the abutment therefore needs to be biofunctionalized to repair the gingival barrier. Here, a mussel-bioinspired implant abutment coating containing tannic acid (TA), cerium and minocycline (TA-Ce-Mino) is reported. TA provides pyrogallol and catechol groups to promote cell adherence. Besides, Ce3+ /Ce4+  conversion exhibits enzyme-mimetic activity to remove reactive oxygen species while generating O2 , therefore promoting anti-inflammatory M2 macrophage polarization to help create a regenerative environment. Minocycline is involved on the TA surface to create local drug storage for responsive antibiosis. Moreover, the underlying therapeutic mechanism is revealed whereby the coating exhibits exogenous antioxidation from the inherent properties of Ce and TA and endogenous antioxidation through mitochondrial homeostasis maintenance and antioxidases promotion. In addition, it stimulates integrin to activate PI3K/Akt and RhoA/ROCK pathways to enhance VEGF-mediated angiogenesis and tissue regeneration. Combining the antibiosis and multidimensional orchestration, TA-Ce-Mino repairs soft tissue barriers and effector cell differentiation, thereby isolating the immune microenvironment from pathogen invasion. Consequently, this study provides critical insight into the design and biological mechanism of abutment surface modification to prevent peri-implantitis.


Assuntos
Peri-Implantite , Humanos , Peri-Implantite/tratamento farmacológico , Peri-Implantite/prevenção & controle , Minociclina , Antioxidantes/farmacologia , Fosfatidilinositol 3-Quinases , Tecido Conjuntivo
7.
J Biomed Mater Res B Appl Biomater ; 110(4): 755-767, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34637601

RESUMO

Human-treated dentin matrix (hTDM) is a biomaterial scaffold, which can induce implant cells to differentiate into odontoblasts and then form neo-dentin. However, hTDM with long storage or prepared by high-speed handpiece would not to form neo-dentin. In this research, we developed two fresh hTDM with different grinding speeds, which were low-speed hTDM (LTDM) with maximum speed of 500 rpm and high-speed hTDM (HTDM) with a speed of 3,80,000 rpm. Here, we aim to understand whether there were induced regeneration capacity differences between LTDM and HTDM. Scanning electron microscope showed that DFCs grew well on both materials, but the morphology of DFCs and the extracellular matrix was different. Especially, the secreted extracellular matrixes on the inner surface of LTDM were regular morphology and ordered arrangement around the dentin tubules. The transcription-quantitative polymerase chain reaction (qRT-PCR), western blot and immunofluorescence assay showed that the dentin markers DSPP and DMP-1 were about 2× greater in DFCs induced by LTDM than by HTDM, and osteogenic marker BSP was about 2× greater in DFCs induced by HTDM than by LTDM. Histological examinations of the harvested grafts observed the formation of neo-tissue were different, and there were neo-dentin formed on the inner surface of LTDM and neo-cementum formed on the outer surface of HTDM. In summary, it found that the induction abilities of LTDM and HTDM are different, and the dentin matrix is directional. This study lays a necessary foundation for searching the key factors of dentin regeneration in future.


Assuntos
Dentina , Matriz Extracelular , Diferenciação Celular , Células Cultivadas , Humanos , Odontoblastos , Regeneração
8.
Front Bioeng Biotechnol ; 10: 1036061, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36324890

RESUMO

Apical periodontitis is a common clinical disease caused by bacteria; bacterial metabolites can cause an imbalance in bone homeostasis, bone mass reduction, and tooth loss. Bone resorption in apical periodontitis causes a concentration of stress in the tooth and periodontal tissues during occlusion, which aggravates the disease. Emerging evidence indicates that bone morphogenetic protein 9 (BMP9), also known as growth differentiation factor 2(Gdf2), may play an important role in tooth and dentoalveolar development. Herein, we investigated the role of BMP9 in the development of apical periodontitis and its effects on the biomechanics of dentoalveolar bone. Apical periodontitis models were established in five BMP9 knockout (KO) mice and five C57BL/6 WT (wild-type) mice. At baseline and 14, 28, and 42 days after modeling, in vivo micro-computed tomography analysis and three-dimensional (3D) reconstruction were performed to evaluate the apical lesion in each mouse, and confirm that the animal models were successfully established. Finite element analysis (FEA) was performed to study the stress and strain at the alveolar fossa of each mouse under the same vertical and lateral stress. FEA revealed that the stress and strain at the alveolar fossa of each mouse gradually concentrated on the tooth cervix. The stress and strain at the tooth cervix gradually increased with time but were decreased at day 42. Under the same lingual loading, the maximum differences of the stress and strain at the tooth root in KO mice were greater than those in WT mice. Thus, these findings demonstrate that BMP9 could affect the biomechanical response of the alveolar fossa at the tooth root in mice with apical periodontitis. Moreover, the effects of BMP9 on the biomechanical response of the alveolar bone may be site-dependent. Overall, this work contributes to an improved understanding of the pathogenesis of apical periodontitis and may inform the development of new treatment strategies for apical periodontitis.

9.
In Vitro Cell Dev Biol Anim ; 57(6): 620-630, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34212339

RESUMO

Human dental follicle cells (HDFCs) are an ideal cell source of stem cells for dental tissue repair and regeneration and they have great potential for regenerative medicine applications. However, the conventional monolayer culture usually reduces cell proliferation and differentiation potential due to the continuous passage during in vitro expansion. In this study, primary HDFC spheroids were generated on 1% agarose, and the HDFCs spontaneously formed cell spheroids in the agarose-coated dishes. Compared with monolayer culture, the spheroid-derived HDFCs exhibited increased proliferative ability for later passage HDFCs as analysed by Cell Counting Kit-8 (CCK-8). The transcription-quantitative polymerase chain reaction (qRT-PCR), western blot and immunofluorescence assay showed that the expression of stemness marker genes Sox2, Oct4 and Nanog was increased significantly in the HDFC spheroids. Furthermore, we found that the odontogenic differentiation capability of HDFCs was significantly improved by spheroid culture in the agarose-coated dishes. On the other hand, the osteogenic differentiation capability was weakened compared with monolayer culture. Our results suggest that spheroid formation of HDFCs in agarose-coated dishes partially restores the proliferative ability of HDFCs at later passages, enhances their stemness and improves odontogenic differentiation capability in vitro. Therefore, spheroid formation of HDFCs has great therapeutic potential for stem cell clinical therapy.


Assuntos
Técnicas de Cultura de Células , Saco Dentário/crescimento & desenvolvimento , Odontogênese/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Saco Dentário/citologia , Saco Dentário/metabolismo , Humanos , Odontogênese/genética , Sefarose/farmacologia , Esferoides Celulares/citologia , Células-Tronco/efeitos dos fármacos
10.
Mater Sci Eng C Mater Biol Appl ; 104: 109908, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31499974

RESUMO

Metals such as Ta (tantalum) and Ti (titanium) have been popularly used as a bone substitute or implants in orthopedic surgery and dentistry, since they have excellent corrosion. For manufacturing porous implants with precise structure, SLM (Selective laser melting), which is one of the 3D (three-dimensional) printing techniques, is always be chosen. To compare biological performances between porous Ta and Ti implants, we designed them with the same porosity, pore shape, pore size, and pore distribution via CAD (computer aided design), and then produced them by SLM. It was shown that the equivalent stress of porous Ta and Ti were 393.62 ±â€¯1.39 MPa and 139.75 ±â€¯14.50 MPa, and their Young's modulus were 3.10 ±â€¯0.03GPa and 5.42 ±â€¯0.07GPa, respectively. Meanwhile, we investigated their biological performance with hBMMSCs (human Bone marrow mesenchymal stem cells) in vitro. The results revealed that both two scaffolds were in favor of hBMMSCs proliferation and osteogenic differentiation. In addition, porous scaffolds were implanted in the femur bone defects rabbits in vivo showed the both porous scaffolds were beneficial to the bone ingrowth and bone-implant fixation. In summary, porous Ta has an equivalent biological performance as traditional porous Ti implants in small bone defect repair. Taken together, porous Ta is a promising material for bone regeneration.


Assuntos
Osseointegração/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Impressão Tridimensional , Tantálio/farmacologia , Alicerces Teciduais/química , Titânio/farmacologia , Ligas , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fluorescência , Humanos , Implantes Experimentais , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Porosidade , Coelhos
11.
J Mech Behav Biomed Mater ; 88: 488-496, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30223212

RESUMO

A properly designed porous scaffold can accelerate the osseointegration process, and the use of computer-aided design (CAD) and additive manufacturing (AM) techniques has the potential to improve the traditional porous scaffold approach. In this study, we evaluate the effect of porous Ti6Al4V (Ti) with different pore structures on osteointegration and osteogenesis. Porous Ti scaffolds with different pore structures based on four commercially available implants were designed and manufactured by CAD and selective laser melting (SLM). Micro-CT showed that SLM was able to produce Ti scaffolds with different pore structures. The mechanical properties evaluated by finite element analysis and compression tests indicated that the four porous scaffolds in our study were mechanically adapted, despite their different mechanical properties. Then, we used 3D-printed porous discs to culture human bone marrow mesenchymal stem cells (hBMMSCs), the main seed cells of bone tissue engineering. The results showed no significant difference among the four groups in cell morphology, viability and proliferation. In addition, four groups showed a comparable mineralization ability even though Ti-g had a higher alkaline phosphatase activity (ALP). In vivo tests in a rabbit model showed that all four groups were suitable for new bone ingrowth and integration. These findings indicate that the four different pore structures in the Ti scaffolds provided good osteointegration and osteogenesis.


Assuntos
Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Fenômenos Mecânicos , Osseointegração/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Impressão Tridimensional , Titânio/química , Ligas , Fenômenos Biomecânicos , Diferenciação Celular/efeitos dos fármacos , Humanos , Teste de Materiais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Porosidade
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