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1.
Methods Mol Biol ; 2206: 223-232, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32754821

RESUMO

Exiting developments in tissue engineering and new insights in stem cell biology have led to new possible strategies for the regeneration of damaged tissues in the oral cavity. The regeneration of the pulp-dentin complex regeneration in particular, has drawn the attention of many researchers because of the high clinical needs. While it is still important to perform in vitro research using a wide variety of cells, scaffolds and growth factors, it is also critical to have a reliable animal model for preclinical trials. In this chapter, we describe a mouse model in which a scaffold resembling a tooth containing dental pulp cells is implanted subcutaneously. We also describe which histological stainings could be used to examine blood vessel formation and the regeneration of the pulp-dentin complex.

2.
J Endod ; 45(4): 427-434, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30833096

RESUMO

INTRODUCTION: Regenerative endodontic procedures (REP) are a novel treatment modality to restore the function of necrotic pulp tissue via stimulation or transplantation of stem cells into the root canal. This study aimed to investigate the immunohistologic outcome of 3 extracted teeth because of sequelae of trauma and unsatisfactory REP outcomes. METHODS: Three immature permanent maxillary central incisors of 3 female patients (6-9 years) were extracted 5.5-22 months after REP. Additionally, 1 sound permanent immature central maxillary incisor of 1 of the included patients was extracted for orthodontic reasons. The teeth were immunohistologically stained with Masson's trichrome, neurofilament (NF), pan cytokeratin, dentin sialophosphoprotein, and Gram+/-. RESULTS: The REP-teeth presented intracanalar vascularized connective/mineralized reparative tissue (RT), which was less organized than the pulp tissue of the sound tooth. Moderate to considerable calcification was observed below the Portland cement used during REP. In 1 case, the RT was NF+; in the 2 other cases, the periodontal ligament and apical granuloma/papilla were NF+. All teeth were Gram+/- negative; nevertheless, inflammatory cells were present in 2 cases. The pan cytokeratin and dentin sialophosphoprotein stainings were not specific enough for 2 cases. CONCLUSIONS: This immunohistologic study of failed REP cases resulted in bacteria-free intracanalar RT and biomaterial-induced calcification. Nevertheless, the presence of inflammatory cells revealed a persistent inflammation. Hence, the clinical and radiographic signs were decisive for tooth survival and multidisciplinary outcome determination.


Assuntos
Falha de Restauração Dentária , Incisivo/lesões , Incisivo/patologia , Endodontia Regenerativa , Calcinose , Criança , Tomografia Computadorizada de Feixe Cônico , Feminino , Humanos , Imuno-Histoquímica , Incisivo/irrigação sanguínea , Incisivo/diagnóstico por imagem , Inflamação , Maxila , Neovascularização Fisiológica , Radiografia Dentária , Estudos Retrospectivos , Células-Tronco , Raiz Dentária/patologia
3.
J Endod ; 45(4): 459-470, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30771897

RESUMO

Despite the fact that external cervical resorption (ECR) is a well-known and rather frequently met condition, the driving force of this phenomenon still remains unclear. Recently, hypoxia has been linked to ECR. Thus, the aim of this work was to investigate the existence of hypoxia in ECR and hypothesize on its role at the time of extraction. This work is a case study of a tooth with ECR. ECR diagnosis was based on clinical and radiographic examination with cone-beam computed tomographic imaging. The extracted tooth was further analyzed by using nanofocus computed tomographic imaging and immunohistology. To investigate the 3-dimensional extent and pattern of ECR, in vivo cone-beam computed tomographic imaging and ex vivo nanofocus computed tomographic imaging were used. Different histologic stains were used to investigate the presence of a hypoxic environment and to gain a better insight into the involved cells, neuronal structures, and remodeling process during ECR. A higher distribution of hypoxia-inducible factor 1a-positive cells was found in the apical part of the resorption area when compared with the coronal area of the resorption. In addition, a similar distribution of hypoxia-inducible factor 1a-positive odontoblasts was observed in the pulp. Three-dimensional analysis of the calcification of the pulp revealed the formation of pulp stones in areas with higher hypoxia. Histology showed that remodeling during ECR can occur according to a layered pattern. This investigation confirms the presence of hypoxia in ECR and shows that there is a gradient of hypoxia within the ECR lesion and surrounding tooth structure. The hypoxic environment within the pulp is also indicated by the formation of pulp stones.


Assuntos
Hipóxia/complicações , Reabsorção da Raiz/etiologia , Reabsorção da Raiz/patologia , Colo do Dente/patologia , Adulto , Calcinose , Tomografia Computadorizada de Feixe Cônico , Polpa Dentária/diagnóstico por imagem , Polpa Dentária/patologia , Humanos , Hipóxia/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Imageamento Tridimensional , Masculino , Radiografia Dentária , Reabsorção da Raiz/diagnóstico por imagem , Ápice Dentário/diagnóstico por imagem , Ápice Dentário/metabolismo , Ápice Dentário/patologia , Colo do Dente/diagnóstico por imagem , Extração Dentária
4.
Sci Rep ; 8(1): 14632, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30279483

RESUMO

Leukocyte- and Platelet-Rich Fibrin (L-PRF) is an autologous platelet concentrate, consisting of a fibrin matrix enriched with platelets, leukocytes and a plethora of cytokines and growth factors. Since L-PRF is produced bedside from whole blood without the use of an anti-coagulant, it is becoming a popular adjuvant in regenerative medicine. While other types of platelet concentrates have been described to stimulate blood vessel formation, little is known about the angiogenic capacities of L-PRF. Therefore, this study aimed to fully characterize the angiogenic potential of L-PRF. With an antibody array, the growth factors released by L-PRF were determined and high levels of CXC chemokine receptor 2 (CXCR-2) ligands and epidermal growth factor (EGF) were found. L-PRF induced in vitro key steps of the angiogenic process: endothelial proliferation, migration and tube formation. In addition, we could clearly demonstrate that L-PRF is able to induce blood vessel formation in vivo, the chorioallantoic membrane assay. In conclusion, we could demonstrate the angiogenic capacity of L-PRF both in vitro and in vivo, underlying the clinical potential of this easy-to-use platelet concentrate.


Assuntos
Neovascularização Fisiológica , Fibrina Rica em Plaquetas , Movimento Celular , Proliferação de Células , Fator de Crescimento Epidérmico , Humanos , Receptores de Interleucina-8B
5.
Circ Heart Fail ; 11(7): e004763, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30002114

RESUMO

BACKGROUND: Large networks of interstitial glycosaminoglycans help to regulate water and electrolyte homeostasis. The relation between dermal interstitial alterations and occurrence of edema in heart failure patients with reduced ejection fraction (HFrEF) is unknown. We hypothesize that in HFrEF patients (1) interstitial glycosaminoglycan density is increased, (2) changes in the interstitial glycosaminoglycan network are associated with interstitial fluid accumulation, and (3) there is a link between the interstitial glycosaminoglycan network and the renin-angiotensin-aldosterone system. METHODS AND RESULTS: Two punch biopsies of the skin were obtained in healthy subjects (n=18) and HFrEF patients (n=29). Alcian blue staining and immunostaining for the angiotensin II type 1 receptor was performed. After obtaining tissue water content, total interstitial glycosaminoglycan (uronic acid) and sulfated glycosaminoglycan were quantified. A venous blood sample, clinical examination, and echocardiography were obtained. A significantly higher interstitial glycosaminoglycan content was observed in HFrEF patients compared with healthy subjects (uronic acid: 13.0±4.2 versus 9.6±1.6 µg/mg; P=0.002; sulfated glycosaminoglycan: 14.1 [11.7; 18.1] versus 10.0 [9.1; 10.8] µg/mg; P<0.001). Uronic acid and sulfated glycosaminoglycan density were strongly associated with tissue water content and peripheral edema (uronic acid: ρ=0.66; P<0.0001 and sulfated glycosaminoglycan: τ=0.58; P<0.0001). Expression of the angiotensin II type 1 receptor was found on dermal cells, although use of angiotensin-converting enzyme inhibitors/angiotensin receptor blocker was associated with significantly lower levels of interstitial glycosaminoglycans in HFrEF patients. CONCLUSIONS: Interstitial glycosaminoglycan concentration is significantly increased in HFrEF patients compared with healthy subjects and correlated with tissue water content and clinical signs of volume overload. A better appreciation of the interstitial compartment might improve management of volume overload in HF.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Insuficiência Cardíaca/tratamento farmacológico , Volume Sistólico/fisiologia , Desequilíbrio Hidroeletrolítico/tratamento farmacológico , Adulto , Idoso , Ecocardiografia/métodos , Feminino , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Sistema Renina-Angiotensina/fisiologia , Volume Sistólico/efeitos dos fármacos , Resultado do Tratamento
6.
Stem Cells Int ; 2018: 9079538, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29535784

RESUMO

Due to the restricted intrinsic capacity of resident chondrocytes to regenerate the lost cartilage postinjury, stem cell-based therapies have been proposed as a novel therapeutic approach for cartilage repair. Moreover, stem cell-based therapies using mesenchymal stem cells (MSCs) or induced pluripotent stem cells (iPSCs) have been used successfully in preclinical and clinical settings. Despite these promising reports, the exact mechanisms underlying stem cell-mediated cartilage repair remain uncertain. Stem cells can contribute to cartilage repair via chondrogenic differentiation, via immunomodulation, or by the production of paracrine factors and extracellular vesicles. But before novel cell-based therapies for cartilage repair can be introduced into the clinic, rigorous testing in preclinical animal models is required. Preclinical models used in regenerative cartilage studies include murine, lapine, caprine, ovine, porcine, canine, and equine models, each associated with its specific advantages and limitations. This review presents a summary of recent in vitro data and from in vivo preclinical studies justifying the use of MSCs and iPSCs in cartilage tissue engineering. Moreover, the advantages and disadvantages of utilizing small and large animals will be discussed, while also describing suitable outcome measures for evaluating cartilage repair.

7.
Stem Cells Int ; 2017: 2582080, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29018483

RESUMO

Adequate vascularization, a restricting factor for the survival of engineered tissues, is often promoted by the addition of stem cells or the appropriate angiogenic growth factors. In this study, human dental pulp stem cells (DPSCs) and stem cells from the apical papilla (SCAPs) were applied in an in vivo model of dental pulp regeneration in order to compare their regenerative potential and confirm their previously demonstrated paracrine angiogenic properties. 3D-printed hydroxyapatite scaffolds containing DPSCs and/or SCAPs were subcutaneously transplanted into immunocompromised mice. After twelve weeks, histological and ultrastructural analysis demonstrated the regeneration of vascularized pulp-like tissue as well as mineralized tissue formation in all stem cell constructs. Despite the secretion of vascular endothelial growth factor in vitro, the stem cell constructs did not display a higher vascularization rate in comparison to control conditions. Similar results were found after eight weeks, which suggests both osteogenic/odontogenic differentiation of the transplanted stem cells and the promotion of angiogenesis in this particular setting. In conclusion, this is the first study to demonstrate the successful formation of vascularized pulp-like tissue in 3D-printed scaffolds containing dental stem cells, emphasizing the promising role of this approach in dental tissue engineering.

8.
J Endod ; 43(9S): S12-S16, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28781091

RESUMO

Dental pulp is a highly vascularized and innervated tissue containing a heterogeneous stem cell population with multilineage differentiation potential. Current endodontic treatments focus on the preservation of the pulp tissue and the regeneration of dental pulp after pathological insults. Human dental pulp stem cells (hDPSCs) are currently investigated as stem cell-based therapy for pulp regeneration and for peripheral nerve injury in which neurons and Schwann cells display limited regenerative capacity. We have developed a neuronal differentiation protocol for hDPSCs that requires neurosphere formation before neuronal maturation. Moreover, Schwann cell differentiation of hDPSCs in our group revealed that differentiated hDPSCs have acquired the ability to myelinate and guide neurites from dorsal root ganglia. Besides their dynamic differentiation capacity, hDPSCs were shown to exert a paracrine effect on neural and endothelial cells. Analysis of hDPSC conditioned medium revealed the secretion of a broad spectrum of growth factors including brain-derived neurotrophic factor, nerve growth factor, vascular endothelial growth factor, and glial-derived neurotrophic factor. Application of the conditioned medium to endothelial cells promoted cell migration and tubulogenesis, indicating a paracrine proangiogenic effect. This hypothesis was enforced by the enhanced formation of blood vessels in the chorioallantoic membrane assay in the presence of hDPSCs. In addition, transplantation of 3-dimensional-printed hydroxyapatite scaffolds containing peptide hydrogels and hDPSCs into immunocompromised mice revealed blood vessel ingrowth, pulplike tissue formation, and osteodentin deposition suggesting osteogenic/odontogenic differentiation of hDPSCs. Future studies in our research group will focus on the pulp regeneration capacity of hDPSCs and the role of fibroblasts within the pulp extracellular matrix.


Assuntos
Polpa Dentária/citologia , Polpa Dentária/fisiologia , Neovascularização Fisiológica , Neurogênese , Células-Tronco/fisiologia , Engenharia Tecidual/métodos , Tecidos Suporte , Animais , Diferenciação Celular , Humanos , Regeneração
9.
PLoS One ; 11(12): e0167807, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27936076

RESUMO

Periodontal ligament stem cells (PDLSCs) represent a good source of multipotent cells for cell-based therapies in regenerative medicine. The success rate of these treatments is severely dependent on the establishment of adequate vasculature in order to provide oxygen and nutrients to the transplanted cells. Pharmacological preconditioning of stem cells has been proposed as a promising method to augment their therapeutic efficacy. In this study, the aim was to improve the intrinsic angiogenic properties of PDLSCs by in vitro pretreatment with deferoxamine (DFX; 100µM), fibroblast growth factor-2 (FGF-2; 10ng/mL) or both substances combined. An antibody array revealed the differential expression of several proteins, including vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). ELISA data confirmed a 1.5 to 1.8-fold increase in VEGF for all tested conditions. Moreover, 48 hours after the removal of DFX, VEGF levels remained elevated (1.8-fold) compared to control conditions. FGF-2 and combination treatment resulted in a 5.4 to 13.1-fold increase in PlGF secretion, whereas DFX treatment had no effect. Furthermore, both PDLSCs as pretreated PDLSCs induced endothelial migration. Despite the significant elevated VEGF levels of pretreated PDLSCs, the induced endothelial migration was not higher by pretreated PDLSCs. We find that the observed induced endothelial cell motility was not dependent on VEGF, since blocking the VEGFR1-3 with Axitinib (0.5nM) did not inhibit endothelial motility towards PDLSCs. Taken together, this study provides evidence that preconditioning with DFX and/or FGF-2 significantly improves the angiogenic secretome of PDLSCs, in particular VEGF and PlGF secretion. However, our data suggest that VEGF is not the only player when it comes to influencing endothelial behavior by the PDLSCs.


Assuntos
Desferroxamina/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Neovascularização Fisiológica , Ligamento Periodontal/citologia , Ligamento Periodontal/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Adolescente , Adulto , Indutores da Angiogênese/farmacologia , Animais , Técnicas de Cultura de Células , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Galinhas , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Humanos , Neovascularização Fisiológica/efeitos dos fármacos , Adulto Jovem
10.
Adv Exp Med Biol ; 951: 199-235, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27837566

RESUMO

Over the past decade, dental tissues have become an attractive source of mesenchymal stem cells (MSCs). Dental stem cells (DSCs) are not only able to differentiate into adipogenic, chondrogenic and osteogenic lineanges, but an increasing amount of research also pointed out their potential applicability in numerous clinical disorders, such as myocardial infarction, neurodegenerative diseases and diabetes. Together with their multilineage differentiation capacity, their easy availability from extracted third molars makes these stem cells a suitable alternative for bone marrow-derived MSCs. More importantly, DSCs appear to retain their stem cell properties following cryopreservation, a key aspect in their long-term preservation and upscale production. However, the vast number of different cryopreservation protocols makes it difficult to draw definite conclusions regarding the behavior of these stem cells. The routine application and banking of DSCs is also associated with some other pitfalls, such as interdonor variability, cell culture-induced changes and the use of animal-derived culture medium additives. Only thorough assessment of these challenges and the implementation of standardized, GMP procedures will successfully lead to better treatment options for patients who no longer benefit from current stem cell therapies.


Assuntos
Bancos de Espécimes Biológicos/organização & administração , Criopreservação/métodos , Polpa Dentária/citologia , Células Secretoras de Insulina/citologia , Miócitos Cardíacos/citologia , Neurônios/citologia , Células-Tronco/citologia , Diferenciação Celular , Proliferação de Células , Crioprotetores/farmacologia , Meios de Cultura/farmacologia , Polpa Dentária/efeitos dos fármacos , Polpa Dentária/fisiologia , Diabetes Mellitus/patologia , Diabetes Mellitus/terapia , Dimetil Sulfóxido/farmacologia , Humanos , Células Secretoras de Insulina/fisiologia , Células Secretoras de Insulina/transplante , Infarto do Miocárdio/patologia , Infarto do Miocárdio/terapia , Miócitos Cardíacos/fisiologia , Miócitos Cardíacos/transplante , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/terapia , Neurônios/fisiologia , Neurônios/transplante , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologia
11.
Stem Cells Int ; 2016: 9762871, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27688777

RESUMO

Within the field of tissue engineering, natural tissues are reconstructed by combining growth factors, stem cells, and different biomaterials to serve as a scaffold for novel tissue growth. As adequate vascularization and innervation are essential components for the viability of regenerated tissues, there is a high need for easily accessible stem cells that are capable of supporting these functions. Within the human tooth and its surrounding tissues, different stem cell populations can be distinguished, such as dental pulp stem cells, stem cells from human deciduous teeth, stem cells from the apical papilla, dental follicle stem cells, and periodontal ligament stem cells. Given their straightforward and relatively easy isolation from extracted third molars, dental stem cells (DSCs) have become an attractive source of mesenchymal-like stem cells. Over the past decade, there have been numerous studies supporting the angiogenic, neuroprotective, and neurotrophic effects of the DSC secretome. Together with their ability to differentiate into endothelial cells and neural cell types, this makes DSCs suitable candidates for dental tissue engineering and nerve injury repair.

12.
Med Res Rev ; 36(6): 1080-1126, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27439773

RESUMO

Stroke is the second most common cause of death and is a major cause of permanent disability. Given the current demographic trend of an ageing population and associated increased risk, the prevalence of and socioeconomic burden caused by stroke will continue to rise. Current therapies are unable to sufficiently ameliorate the disease outcome and are not applicable to all patients. Therefore, strategies such as cell-based therapies with mesenchymal stem cell (MSC) or induced pluripotent stem cell (iPSC) pave the way for new treatment options for stroke. These cells showed great preclinical promise despite the fact that the precise mechanism of action and the optimal administration route are unknown. To gain dynamic insights into the underlying repair processes after stem cell engraftment, noninvasive imaging modalities were developed to provide detailed spatial and functional information on the donor cell fate and host microenvironment. This review will focus on MSCs and iPSCs as types of widely used stem cell sources in current (bio)medical research and compare their efficacy and potential to ameliorate the disease outcome in animal stroke models. In addition, novel noninvasive imaging strategies allowing temporospatial in vivo tracking of transplanted cells and coinciding evaluation of neuronal repair following stroke will be discussed.


Assuntos
Isquemia Encefálica/terapia , Transplante de Células-Tronco/métodos , Acidente Vascular Cerebral/terapia , Animais , Isquemia Encefálica/diagnóstico por imagem , Isquemia Encefálica/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/transplante , Medições Luminescentes/métodos , Imagem por Ressonância Magnética/métodos , Transplante de Células-Tronco Mesenquimais/métodos , Tomografia por Emissão de Pósitrons/métodos , Regeneração/fisiologia , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/patologia , Tomografia Computadorizada de Emissão de Fóton Único/métodos
13.
Clin Oral Investig ; 20(4): 807-14, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26250796

RESUMO

OBJECTIVES: An immunohistological study of an infected immature permanent human tooth after a regenerative endodontic procedure (REP) was conducted in order to determine the histologic outcome of this procedure. Besides observed signs of angiogenesis and neurogenesis, repair and/or regeneration of the pulp-dentin complex was also investigated. MATERIALS AND METHODS: A REP was performed on tooth 45 of a 10-year-old girl. Eleven months post-treatment, the tooth had to be removed for orthodontic reasons. The following investigations were performed: immunohistology and radiographic quantification of root development. After hematoxylin-eosin (HE) staining, the following immunomarkers were selected: neurofilament (NF), pan cytokeratin (PK), osteocalcin (OC), and CD34. RESULTS: The REP resulted in clinical and radiographic healing of the periradicular lesion and quantifiable root development. The HE staining matches with the medical imaging post-REP: underneath the mineral trioxide aggregate a calcified bridge with cell inclusions, connective pulp-like tissue (PLT) with blood vessels, osteodentin against the root canal walls, on the root surface cementum (Ce), and periodontal ligament (PDL). The PDL was PK(+). The blood vessels in the PLT and PDL were CD34(+). The Ce, osteodentin, and stromal cells in the PLT were OC(+). The neurovascular bundles in the PLT were NF(+). CONCLUSIONS: Immunohistologically, REP of this infected immature permanent tooth resulted in an intracanalar connective tissue with a regulated physiology, but not pulp tissue. CLINICAL RELEVANCE: REP of an immature permanent infected tooth may heal the periapical infection and may result in a combination of regeneration and repair of the pulp-dentin complex.


Assuntos
Apexificação , Necrose da Polpa Dentária , Ápice Dentário , Polpa Dentária , Feminino , Humanos , Raiz Dentária
14.
Stem Cells Dev ; 24(14): 1610-22, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-25869156

RESUMO

Although regenerative endodontic procedures have yielded an impressive body of favorable outcomes, the treatment of necrotic immature permanent teeth in particular remains to be a challenge. Recent advances in dental stem cell (DSC) research have gained increasing insight in their regenerative potential and prospective use in the formation of viable dental tissues. Numerous studies have already reported successful dental pulp regeneration following application of dental pulp stem cells, stem cells from the apical papilla, or dental follicle precursor cells in different in vivo models. Next to responsive cells, dental tissue engineering also requires the support of an appropriate scaffold material, ranging from naturally occurring polymers to treated dentin matrix components. However, the routine use and banking of DSCs still holds some major challenges, such as culture-associated differences, patient-related variability, and the effects of culture medium additives. Only in-depth evaluation of these problems and the implementation of standardized models and protocols will effectively lead to better alternatives for patients who no longer benefit from current treatment protocols.


Assuntos
Polpa Dentária/citologia , Regeneração Tecidual Guiada/métodos , Células-Tronco/citologia , Engenharia Tecidual/métodos , Papila Dentária/citologia , Saco Dentário/citologia , Endodontia/métodos , Humanos , Neovascularização Fisiológica , Tecidos Suporte
15.
Stem Cells Dev ; 24(3): 296-311, 2015 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-25203005

RESUMO

Cell-based therapies are emerging as an alternative treatment option to promote functional recovery in patients suffering from neurological disorders, which are the major cause of death and permanent disability. The present study aimed to differentiate human dental pulp stem cells (hDPSCs) toward functionally active neuronal cells in vitro. hDPSCs were subjected to a two-step protocol. First, neuronal induction was acquired through the formation of neurospheres, followed by neuronal maturation, based on cAMP and neurotrophin-3 (NT-3) signaling. At the ultrastructural level, it was shown that the intra-spheral microenvironment promoted intercellular communication. hDPSCs grew out of the neurospheres in vitro and established a neurogenic differentiated hDPSC culture (d-hDPSCs) upon cAMP and NT-3 signaling. d-hDPSCs were characterized by the increased expression of neuronal markers such as neuronal nuclei, microtubule-associated protein 2, neural cell adhesion molecule, growth-associated protein 43, synapsin I, and synaptophysin compared with nondifferentiated hDPSCs. Enzyme-linked immunosorbent assay demonstrated that the secretion of brain-derived neurotrophic factor, vascular endothelial growth factor, and nerve growth factor differed between d-hDPSCs and hDPSCs. d-hDPSCs acquired neuronal features, including multiple intercommunicating cytoplasmic extensions and increased vesicular transport, as shown by the electron microscopic observation. Patch clamp analysis demonstrated the functional activity of d-hDPSCs by the presence of tetrodotoxin- and tetraethyl ammonium-sensitive voltage-gated sodium and potassium channels, respectively. A subset of d-hDPSCs was able to fire a single action potential. The results reported in this study demonstrate that hDPSCs are capable of neuronal commitment following neurosphere formation, characterized by distinct morphological and electrophysiological properties of functional neuronal cells.


Assuntos
Polpa Dentária/citologia , Neurogênese , Neurônios/citologia , Potenciais de Ação , Adolescente , Adulto , Comunicação Celular , Técnicas de Cultura de Células , Separação Celular/métodos , Forma Celular , Células Cultivadas , Microambiente Celular , Meios de Cultura/farmacologia , Feminino , Humanos , Canais Iônicos/fisiologia , Masculino , Microscopia Eletrônica , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Técnicas de Patch-Clamp , Esferoides Celulares , Adulto Jovem
16.
Pharmacol Ther ; 143(2): 181-96, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24594234

RESUMO

Mesenchymal stem cells or multipotent stromal cells (MSCs) have initially captured attention in the scientific world because of their differentiation potential into osteoblasts, chondroblasts and adipocytes and possible transdifferentiation into neurons, glial cells and endothelial cells. This broad plasticity was originally hypothesized as the key mechanism of their demonstrated efficacy in numerous animal models of disease as well as in clinical settings. However, there is accumulating evidence suggesting that the beneficial effects of MSCs are predominantly caused by the multitude of bioactive molecules secreted by these remarkable cells. Numerous angiogenic factors, growth factors and cytokines have been discovered in the MSC secretome, all have been demonstrated to alter endothelial cell behavior in vitro and induce angiogenesis in vivo. As a consequence, MSCs have been widely explored as a promising treatment strategy in disorders caused by insufficient angiogenesis such as chronic wounds, stroke and myocardial infarction. In this review, we will summarize into detail the angiogenic factors found in the MSC secretome and their therapeutic mode of action in pathologies caused by limited blood vessel formation. Also the application of MSC as a vehicle to deliver drugs and/or genes in (anti-)angiogenesis will be discussed. Furthermore, the literature describing MSC transdifferentiation into endothelial cells will be evaluated critically.


Assuntos
Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica/fisiologia , Animais , Doenças Cardiovasculares/fisiopatologia , Diferenciação Celular/fisiologia , Transdiferenciação Celular/fisiologia , Citocinas/metabolismo , Células Endoteliais/metabolismo , Técnicas de Transferência de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Transplante de Células-Tronco Mesenquimais , Neoplasias/fisiopatologia
17.
PLoS One ; 8(8): e71104, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23951091

RESUMO

Angiogenesis, the formation of capillaries from pre-existing blood vessels, is a key process in tissue engineering. If blood supply cannot be established rapidly, there is insufficient oxygen and nutrient transport and necrosis of the implanted tissue will occur. Recent studies indicate that the human dental pulp contains precursor cells, named dental pulp stem cells (hDPSC) that show self-renewal and multilineage differentiation capacity. Since these cells can be easily isolated, cultured and cryopreserved, they represent an attractive stem cell source for tissue engineering. Until now, only little is known about the angiogenic abilities and mechanisms of the hDPSC. In this study, the angiogenic profile of both cell lysates and conditioned medium of hDPSC was determined by means of an antibody array. Numerous pro-and anti-angiogenic factors such as vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1), plasminogen activator inhibitor-1 (PAI-1) and endostatin were found both at the mRNA and protein level. hDPSC had no influence on the proliferation of the human microvascular endothelial cells (HMEC-1), but were able to significantly induce HMEC-1 migration in vitro. Addition of the PI3K-inhibitor LY294002 and the MEK-inhibitor U0126 to the HMEC-1 inhibited this effect, suggesting that both Akt and ERK pathways are involved in hDPSC-mediated HMEC-1 migration. Antibodies against VEGF also abolished the chemotactic actions of hDPSC. Furthermore, in the chicken chorioallantoic membrane (CAM) assay, hDPSC were able to significantly induce blood vessel formation. In conclusion, hDPSC have the ability to induce angiogenesis, meaning that this stem cell population has a great clinical potential, not only for tissue engineering but also for the treatment of chronic wounds, stroke and myocardial infarctions.


Assuntos
Indutores da Angiogênese/metabolismo , Polpa Dentária/citologia , Neovascularização Fisiológica/fisiologia , Células-Tronco/metabolismo , Adolescente , Indutores da Angiogênese/farmacologia , Animais , Butadienos/farmacologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Cromonas/farmacologia , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Endostatinas/genética , Endostatinas/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Imuno-Histoquímica , Morfolinas/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Nitrilos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adulto Jovem
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