RESUMO
Fibroblast growth factor 2 (FGF2) is a crucial factor in odontoblast differentiation and dentin matrix deposition, which facilitates pulpodentin repair and regeneration. Nevertheless, the specific biological function of FGF2 in odontoblastic differentiation remains unclear because it is controlled by complex signalling pathways. This study aimed to investigate the mechanism underlying the effect of FGF2 on osteo/odontogenic differentiation of stem cells from the apical papilla (SCAP). SCAP were pretreated with conditioned media containing FGF2 for 1 week, followed by culturing in induced differentiation medium for another week. RNA sequencing (RNA-seq) combined with quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to evaluate the pathways affected by FGF2 in SCAP. Osteo/odontogenic differentiation of SCAP was determined using Alizarin red S staining, alkaline phosphatase staining, RT-qPCR, and western blotting. Pretreatment with FGF2 for 1 week increased the osteo/odontogenic differentiation ability of SCAP. RNA-seq and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that phosphatidylinositol 3-kinase (PI3K)/AKT signalling is involved in the osteogenic function of FGF2. RT-qPCR results indicated that SCAP expressed FGF receptors, and western blotting showed that p-AKT was reduced in FGF2-pretreated SCAP. The activation of the PI3K/AKT pathway partially reversed the stimulatory effect of FGF2 on osteo/odontogenic differentiation of SCAP. Our findings suggest that pretreatment with FGF2 enhances the osteo/odontogenic differentiation ability of SCAP by inhibiting the PI3K/AKT pathway.
Assuntos
Diferenciação Celular , Papila Dentária , Fator 2 de Crescimento de Fibroblastos , Odontogênese , Osteogênese , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Células-Tronco , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Diferenciação Celular/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Papila Dentária/citologia , Papila Dentária/metabolismo , Humanos , Odontogênese/efeitos dos fármacos , Células-Tronco/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Células CultivadasRESUMO
Skeletal muscle satellite cells (SCs) are essential for muscle regeneration. Their proliferation and differentiation are influenced by fibroblast growth factor (FGF)-2. In this study, we screened for FGF-2-derived peptides that promote SC proliferation. Utilizing photocleavable peptide array technology, a library of 7-residue peptides was synthesized, and its effect on SC proliferation was examined using a mixture of five peptides. The results showed that peptides 1-5 (136%), 21-25 (136%), 26-30 (141%), 31-35 (159%), 71-75 (135%), 76-80 (144%), and 126-130 (137%) significantly increased SC proliferation. Further experiments revealed that peptide 33, CKNGGFF, enhanced SC proliferation. Furthermore, its extended form, peptide 33-13, CKNGGFFLRIHPD, promoted SC proliferation and increased the percentage of Pax7-positive cells, indicating that SCs were maintained in an undifferentiated state. The addition of FGF-2 and peptide 33-13 further induced cell proliferation but did not increase the percentage of Pax7-positive cells. A proliferation assay using an FGF receptor (FGFR) inhibitor suggested that peptide 33-13 acts through the FGFR-mediated and other pathways. Although further research is necessary to explore the mechanisms of action of these peptides and their potential for in vivo and in vitro use, the high sequence conservation of peptides 33 and 33-13 in FGF-2 across multiple species suggests their broad application prospects in biomedical engineering and biotechnology.
Assuntos
Proliferação de Células , Fator 2 de Crescimento de Fibroblastos , Peptídeos , Células Satélites de Músculo Esquelético , Animais , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Camundongos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células CultivadasRESUMO
The homeostasis of human pluripotent stem cells (hPSCs) requires the signaling balance of extracellular factors. Exogenous regulators from cell culture medium have been widely reported, but little attention has been paid to the autocrine factor from hPSCs themselves. In this report, we demonstrate that extracellular signal-related kinase 5 (ERK5) regulates endogenous autocrine factors essential for pluripotency and differentiation. ERK5 inhibition leads to erroneous cell fate specification in all lineages even under lineage-specific induction. hPSCs can self-renew under ERK5 inhibition in the presence of fibroblast growth factor 2 (FGF2) and transforming growth factor ß (TGF-ß), although NANOG expression is partially suppressed. Further analysis demonstrates that ERK5 promotes the expression of autocrine factors such as NODAL, FGF8, and WNT3. The addition of NODAL protein rescues NANOG expression and differentiation phenotypes under ERK5 inhibition. We demonstrate that constitutively active ERK5 pathway allows self-renewal even without essential growth factors FGF2 and TGF-ß. This study highlights the essential contribution of autocrine pathways to proper maintenance and differentiation.
Assuntos
Comunicação Autócrina , Proteína Quinase 7 Ativada por Mitógeno , Proteína Homeobox Nanog , Células-Tronco Pluripotentes , Humanos , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Proliferação de Células , Autorrenovação Celular , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/genética , Proteína Homeobox Nanog/metabolismo , Proteína Homeobox Nanog/genética , Proteína Nodal/metabolismo , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismoRESUMO
The healing of severe chronic skin wounds in chronic diabetic patients is still a huge clinical challenge due to complex regeneration processes and control signals. Therefore, a single approach is difficult in obtaining satisfactory therapeutic efficacy for severe diabetic skin wounds. In this study, we adopted a composite strategy for diabetic skin wound healing. First, we fabricated a collagen-based biomimetic skin scaffold. The human basic fibroblast growth factor (bFGF) gene was electrically transduced into human umbilical cord mesenchymal stromal cells (UC-MSCs), and the stable bFGF-overexpressing UC-MSCs (bFGF-MSCs) clones were screened out. Then, an inspired collagen scaffold loaded with bFGF-MSCs was applied to treat full-thickness skin incision wounds in a streptozotocin-induced diabetic rat model. The mechanism of skin damage repair in diabetes mellitus was investigated using RNA-Seq and Western blot assays. The bioinspired collagen scaffold demonstrated good biocompatibility for skin-regeneration-associated cells such as human fibroblast (HFs) and endothelial cells (ECs). The bioinspired collagen scaffold loaded with bFGF-MSCs accelerated the diabetic full-thickness incision wound healing including cell proliferation enhancement, collagen deposition, and re-epithelialization, compared with other treatments. We also showed that the inspired skin scaffold could enhance the in vitro tube formation of ECs and the early angiogenesis process of the wound tissue in vivo. Further findings revealed enhanced angiogenic potential in ECs stimulated by bFGF-MSCs, evidenced by increased AKT phosphorylation and elevated HIF-1α and HIF-1ß levels, indicating the activation of HIF-1 pathways in diabetic wound healing. Based on the superior biocompatibility and bioactivity, the novel bioinspired skin healing materials composed of the collagen scaffold and bFGF-MSCs will be promising for healing diabetic skin wounds and even other refractory tissue regenerations. The bioinspired collagen scaffold loaded with bFGF-MSCs could accelerate diabetic wound healing via neovascularization by activating HIF-1 pathways.
Assuntos
Colágeno , Diabetes Mellitus Experimental , Fator 2 de Crescimento de Fibroblastos , Células-Tronco Mesenquimais , Neovascularização Fisiológica , Transdução de Sinais , Pele , Alicerces Teciduais , Cicatrização , Humanos , Cicatrização/efeitos dos fármacos , Animais , Células-Tronco Mesenquimais/metabolismo , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/farmacologia , Colágeno/química , Ratos , Alicerces Teciduais/química , Pele/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Ratos Sprague-Dawley , Masculino , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fator 1 Induzível por Hipóxia/metabolismoRESUMO
Mesenchymal stromal cells (MSCs) have the potential to be used as autologous or allogenic cell therapy in several diseases due to their beneficial secretome and capacity for immunomodulation and differentiation. However, clinical trials using MSCs require a large number of cells. As an alternative to traditional culture flasks, suspension bioreactors provide a scalable platform to produce clinically relevant quantities of cells. When cultured in bioreactors, anchorage-dependent cells like MSCs require the addition of microcarriers, which provide a surface for cell attachment while in suspension. The best performing microcarriers are typically coated in animal derived proteins, which increases cellular attachment and proliferation but present issues from a regulatory perspective. To overcome this issue, a recombinant fusion protein was generated linking basic fibroblast growth factor (bFGF) to a cellulose-specific carbohydrate binding module (CBM) and used to functionalize the surface of cellulose microcarriers for the expansion of human umbilical MSCs in suspension bioreactors. The fusion protein was shown to support the growth of MSCs when used as a soluble growth factor in the absence of cellulose, readily bound to cellulose microcarriers in a dose-dependent manner, and ultimately improved the expansion of MSCs when grown in bioreactors using cellulose microcarriers. The use of CBM fusion proteins offers a simple method for the surface immobilization of growth factors to animal component-free substrates such as cellulose, which can be used alongside bioreactors to increase growth factor lifespan, decrease culture medium cost, and increase cell production in the manufacturing of therapeutic cells.
Assuntos
Reatores Biológicos , Proliferação de Células , Celulose , Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Humanos , Celulose/química , Proliferação de Células/efeitos dos fármacos , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/química , Tamanho da Partícula , Teste de Materiais , Células Cultivadas , Técnicas de Cultura de Células , Módulos de Ligação de CarboidratosRESUMO
OBJECTIVES: This study evaluated the effect of intratracheal administration of basic fibroblast growth factor (bFGF) on tracheal healing following implantation of a novel layered polyglycolic acid (PGA) material to replace a critical-size defect in rat trachea. METHODS: A critical-size defect in the rat cervical trachea was covered with PGA. Distilled water (DW) or 3.125, 6.25, 12.5 or 25 µg bFGF was administered into the trachea for 2 weeks (n = 6 for each of 5 groups). Regenerated areas of cilia, ciliary beat frequency and ciliary transport function (CTF) in the centre of the PGA were measured. To examine potential side effects of intratracheal administration of bFGF, the right lower lobe was pathologically evaluated. RESULTS: All rats survived during the study period. Histological examination showed ciliated epithelization on the PGA material after 2 weeks. Bronchoscopy revealed stenosis due to granulation following administration of high concentrations of bFGF (12.5 and 25 µg). Compared with the DW group, groups administered 3.125, 6.25, 12.5 and 25 µg bFGF had significantly larger areas of regenerated cilia (15.2%, 27.0%, 41.3%, 33.1% and 31.0%, respectively; P = 0.00143), improved ciliary beat frequency (7.10, 8.18, 10.10, 9.50 and 9.50 Hz, respectively), and improved CTS (6.40, 9.54, 16.89, 16.41 and 14.29 µm/sec, respectively). Pathological examination of the right lower lobe revealed pulmonary fibrosis and hyperplasia with high concentrations of bFGF (12.5 and 25 µg). CONCLUSIONS: Intratracheal administration of bFGF effectively promoted tracheal regeneration at an optimal dose of 6.25 µg following implantation of an artificial trachea.
Assuntos
Fator 2 de Crescimento de Fibroblastos , Traqueia , Cicatrização , Animais , Traqueia/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/administração & dosagem , Fator 2 de Crescimento de Fibroblastos/farmacologia , Ratos , Masculino , Cicatrização/efeitos dos fármacos , Ratos Sprague-Dawley , Órgãos Artificiais , Cílios/efeitos dos fármacosRESUMO
The direct antitumor effect of bevacizumab (BEV) has long been debated. Evidence of the direct antitumor activities of drugs are mainly obtained from in vitro experiments, which are greatly affected by experimental conditions. In this study, we evaluated the effect of BEV-containing medium renewal on the results of in vitro cytotoxicity experiments in A549 and U251 cancer cells. We observed starkly different results between the experiments with and without BEV-containing medium renewal. Specifically, BEV inhibited the tumor cell growth in the timely replacement with a BEV-containing medium but promoted tumor cell growth without medium renewal. Meanwhile, compared with the control, a significant basic fibroblast growth factor (bFGF) accumulation in the supernatant was observed in the group without medium renewal but none in that with replaced medium. Furthermore, bFGF neutralization partially reversed the pro-proliferative effect of BEV in the medium non-renewed group, while exogenous bFGF attenuated the tumor cell growth inhibition of BEV in the medium-renewed group. Our data explain the controversy over the direct antitumor effect of BEV in different studies from the perspective of the compensatory autocrine cytokines in tumor cells.
Assuntos
Bevacizumab , Proliferação de Células , Fator 2 de Crescimento de Fibroblastos , Humanos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Bevacizumab/farmacologia , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Meios de Cultura/química , Meios de Cultura/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Inibidores da Angiogênese/farmacologia , Células A549 , Antineoplásicos Imunológicos/farmacologiaRESUMO
Traumatic brain injury (TBI) and spinal cord injury (SCI) are neurological conditions that result from immediate mechanical injury, as well as delayed injury caused by local inflammation. Furthermore, TBI and SCI often lead to secondary complications, including pressure wounds of the skin, which can heal slowly and are prone to infection. Pressure wounds are localized areas of damaged tissue caused by prolonged pressure on the skin due to immobility and loss of neurological sensation. With the aim to ameliorate these symptoms, we investigated whether fibroblast growth factors 2 (FGF-2) could contribute to recovery. FGF-2 plays a significant role in both neurogenesis and skin wound healing. We developed a recombinant fusion protein containing FGF-2 linked to elastin-like polypeptides (FGF-ELP) that spontaneously self-assembles into nanoparticles at around 33 °C. The nanoparticle's size was ranging between 220 and 250 nm in diameter at 2 µM. We tested this construct for its ability to address neuronal and skin cell injuries. Hydrogen peroxide was used to induce oxidant-mediated injury on cultured neuronal cells to mimic the impact of reactive oxidants released during the inflammatory response in vivo. We found that FGF-ELP nanoparticles protected against hydrogen peroxide-mediated injury and promoted neurite outgrowth. In the skin cell models, cells were depleted from serum to mimic the reduced levels of nutrients and growth factors in chronic skin wounds. FGF-ELP increased the proliferation and migration of human keratinocytes, fibroblasts, and endothelial cells. FGF-ELP is, therefore, a potentially useful agent to provide both neuroprotection and promotion of cellular processes involved in skin wound healing.
Assuntos
Nanopartículas , Neurônios , Pele , Animais , Humanos , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Peróxido de Hidrogênio/farmacologia , Teste de Materiais , Nanopartículas/química , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurônios/metabolismo , Oxidantes/química , Oxidantes/farmacologia , Tamanho da Partícula , Pele/patologia , Pele/efeitos dos fármacos , Cicatrização/efeitos dos fármacosRESUMO
Guided bone regeneration (GBR) is currently the most widely used bone augmentation technique in oral clinics. However, infection and soft tissue management remain the greatest challenge. In this study, a Janus sponge/electrospun fibre membrane containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and chlorhexidine (CHX) were prepared to optimize its application as a barrier membrane for GBR. The loose sponge part was covalently bonded with the fiber part which has a dense structure. The composed scaffold exhibited superior biocompatibility and antibacterial activity verified by in vitro test. A rat model of unilateral skull bone injury was used to confirm the effectiveness on both hard and soft tissue regeneration. The chitosan sponge on the soft tissue side containing EGF, bFGF and CHX had a loose structure, promoting collagen and cell regeneration and exerting an antibacterial effect. Meanwhile, the dense PLGA/PCL layer on the hard tissue side prevented fibroblast entry into the bone defect, thereby facilitating bone regeneration. The Janus composite scaffold provides a promising strategy for oral tissue restoration.
Assuntos
Regeneração Óssea , Clorexidina , Fator de Crescimento Epidérmico , Fator 2 de Crescimento de Fibroblastos , Animais , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/química , Regeneração Óssea/efeitos dos fármacos , Ratos , Fator de Crescimento Epidérmico/farmacologia , Fator de Crescimento Epidérmico/química , Clorexidina/farmacologia , Clorexidina/química , Ratos Sprague-Dawley , Antibacterianos/farmacologia , Antibacterianos/química , Alicerces Teciduais/química , Masculino , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Regeneração Tecidual Guiada/métodos , Quitosana/química , Quitosana/farmacologiaRESUMO
Bile duct injury presents a significant clinical challenge following hepatobiliary surgery, necessitating advancements in the repair of damaged bile ducts is a persistent issue in biliary surgery. 3D printed tubular scaffolds have emerged as a promising approach for the repair of ductal tissues, yet the development of scaffolds that balance exceptional mechanical properties with biocompatibility remains an ongoing challenge. This study introduces a novel, bio-fabricated bilayer bile duct scaffold using a 3D printing technique. The scaffold comprises an inner layer of polyethylene glycol diacrylate (PEGDA) to provide high mechanical strength, and an outer layer of biocompatible, methacryloylated recombinant collagen type III (rColMA) loaded with basic fibroblast growth factor (bFGF)-encapsulated liposomes (bFGF@Lip). This design enables the controlled release of bFGF, creating an optimal environment for the growth and differentiation of bone marrow mesenchymal stem cells (BMSCs) into cholangiocyte-like cells. These cells are instrumental in the regeneration of bile duct tissues, evidenced by the pronounced expression of cholangiocyte differentiation markers CK19 and CFTR. The PEGDA//rColMA/bFGF@Lip bilayer bile duct scaffold can well simulate the bile duct structure, and the outer rColMA/bFGF@Lip hydrogel can well promote the growth and differentiation of BMSCs into bile duct epithelial cells. In vivo experiments showed that the scaffold did not cause cholestasis in the body. This new in vitro pre-differentiated active 3D printed scaffold provides new ideas for the study of bile duct tissue replacement.
Assuntos
Ductos Biliares , Diferenciação Celular , Hidrogéis , Células-Tronco Mesenquimais , Polietilenoglicóis , Impressão Tridimensional , Polietilenoglicóis/química , Hidrogéis/química , Hidrogéis/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Colágeno/química , Alicerces Teciduais/química , Camundongos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Células Cultivadas , Humanos , MasculinoRESUMO
Once damaged, cartilage has poor intrinsic capacity to repair itself. Current cartilage repair strategies cannot restore the damaged tissue sufficiently. It is hypothesized that biomimetic scaffolds, which can recapitulate important properties of the cartilage extracellular matrix, play a beneficial role in supporting cell behaviors such as growth, cartilage differentiation, and integration with native cartilage, ultimately facilitating tissue recovery. Adipose-derived stem cells regenerated cartilage upon the sequential release of transforming growth factor ß1(TGFß1) and fibroblast growth factor 2(FGF2) using a nanofibrous scaffold, in order to get the recovery of functional cartilage. Experiments in vitro have demonstrated that the release sequence of growth factors FGF2 to TGFß1 is the most essential to promote adipose-derived stem cells into chondrocytes that then synthesize collagen II. Mouse subcutaneous implantation indicated that the treatment sequence of FGF2 to TGFß1 was able to significantly induce multiple increase in cartilage regeneration in vivo. This result demonstrates that the group treated with FGF2 to TGFß1 released from a nanofibrous scaffold provides a good strategy for cartilage regeneration by making a favorable microenvironment for cell growth and cartilage regeneration.
Assuntos
Diferenciação Celular , Fator 2 de Crescimento de Fibroblastos , Nanofibras , Células-Tronco , Alicerces Teciduais , Fator de Crescimento Transformador beta1 , Animais , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Camundongos , Nanofibras/química , Diferenciação Celular/efeitos dos fármacos , Alicerces Teciduais/química , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologia , Condrogênese/efeitos dos fármacos , Cartilagem/efeitos dos fármacos , Cartilagem/citologia , Cartilagem/fisiologia , Tecido Adiposo/citologia , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/fisiologia , Células Cultivadas , Engenharia Tecidual/métodosRESUMO
Decellularized extracellular matrix hydrogel (ECM hydrogel), a natural material derived from normal tissue with unique biocompatibility properties, is widely used for tissue repair. However, there are still problems such as poor biological activity and insufficient antimicrobial property. To overcome these drawbacks, fibroblast growth factor 2 (FGF 2) containing exosome (exoFGF 2) was prepared to increase the biological activity. Furthermore, the antimicrobial capacity of ECM hydrogel was optimised by using copper ions as a ligand-bonded cross-linking agent. The decellularized extracellular matrix hydrogel, intricately cross-linked with copper ions through ligand bonds and loaded with FGF 2 containing exosome (exoFGF 2@ECM/Cu2+ hydrogel), has demonstrated exceptional biocompatibility and antimicrobial properties. In vitro, exoFGF 2@ECM/Cu2+ hydrogel effectively promoted cell proliferation, migration, antioxidant and inhibited bacterial growth. In vivo, the wound area of rat treated with exoFGF 2@ECM/Cu2+ hydrogels were significantly smaller than that of other groups at Day 5 (45.24% ± 3.15%), Day 10 (92.20% ± 2.31%) and Day 15 (95.22% ± 1.28%). Histological examination showed that exoFGF 2@ECM/Cu2+ hydrogels promoted angiogenesis and collagen deposition. Overall, this hydrogel has the potential to inhibit bacterial growth and effectively promote wound healing in a variety of clinical applications.
Assuntos
Proliferação de Células , Exossomos , Matriz Extracelular , Fator 2 de Crescimento de Fibroblastos , Hidrogéis , Pele , Cicatrização , Hidrogéis/química , Hidrogéis/farmacologia , Animais , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/química , Exossomos/química , Exossomos/metabolismo , Ratos , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Cicatrização/efeitos dos fármacos , Pele/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ratos Sprague-Dawley , Humanos , Cobre/química , Cobre/farmacologia , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Masculino , Camundongos , Movimento Celular/efeitos dos fármacos , Engenharia Tecidual/métodosRESUMO
Dysfunction of pancreatic δ cells contributes to the etiology of diabetes. Despite their important role, human δ cells are scarce, limiting physiological studies and drug discovery targeting δ cells. To date, no directed δ-cell differentiation method has been established. Here, we demonstrate that fibroblast growth factor (FGF) 7 promotes pancreatic endoderm/progenitor differentiation, whereas FGF2 biases cells towards the pancreatic δ-cell lineage via FGF receptor 1. We develop a differentiation method to generate δ cells from human stem cells by combining FGF2 with FGF7, which synergistically directs pancreatic lineage differentiation and modulates the expression of transcription factors and SST activators during endoderm/endocrine precursor induction. These δ cells display mature RNA profiles and fine secretory granules, secrete somatostatin in response to various stimuli, and suppress insulin secretion from in vitro co-cultured ß cells and mouse ß cells upon transplantation. The generation of human pancreatic δ cells from stem cells in vitro would provide an unprecedented cell source for drug discovery and cell transplantation studies in diabetes.
Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes , Humanos , Animais , Camundongos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Somatostatina/metabolismo , Células Secretoras de Somatostatina/citologia , Endoderma/citologia , Endoderma/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Pâncreas/citologia , Pâncreas/metabolismo , Somatostatina/metabolismo , Linhagem da Célula , Insulina/metabolismo , Secreção de InsulinaRESUMO
In recent years, the interest in cell transplantation therapy using human dental pulp cells (DPCs) has been increasing. However, significant differences exist in the individual cellular characteristics of human DPC clones and in their therapeutic efficacy in rodent models of spinal cord injury (SCI); moreover, the cellular properties associated with their therapeutic efficacy for SCI remain unclear. Here, using DPC clones from seven different donors, we found that most of the clones were highly resistant to H2O2 cytotoxicity if, after transplantation, they significantly improved the locomotor function of rats with complete SCI. Therefore, we examined the effects of the basic fibroblast growth factor 2 (FGF2) and bardoxolone methyl (RTA402), which is a nuclear factor erythroid 2-related factor 2 (Nrf2) chemical activator, on the total antioxidant capacity (TAC) and the resistance to H2O2 cytotoxicity. FGF2 treatment enhanced the resistance of a subset of clones to H2O2 cytotoxicity. Regardless of FGF2 priming, RTA402 markedly enhanced the resistance of many DPC clones to H2O2 cytotoxicity, concomitant with the upregulation of heme oxygenase-1 (HO-1) and NAD(P)H-quinone dehydrogenase 1 (NQO1). With the exception of a subset of clones, the TAC was not increased by either FGF2 priming or RTA402 treatment alone, whereas it was significantly upregulated by both treatments in each clone, or among all seven DPC clones together. Thus, the TAC and resistance to H2O2 cytotoxicity were, to some extent, independently regulated and were strongly enhanced by both FGF2 priming and RTA402 treatment. Moreover, even a DPC clone that originally exhibited no therapeutic effect on SCI improved the locomotor function of mice with SCI after transplantation under both treatment regimens. Thus, combined with FGF2, RTA402 may increase the number of transplanted DPCs that migrate into and secrete neurotrophic factors at the lesion epicenter, where reactive oxygen species are produced at a high level.
Assuntos
Antioxidantes , Polpa Dentária , Fator 2 de Crescimento de Fibroblastos , Fator 2 Relacionado a NF-E2 , Traumatismos da Medula Espinal , Polpa Dentária/metabolismo , Polpa Dentária/citologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Humanos , Traumatismos da Medula Espinal/terapia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/tratamento farmacológico , Ratos , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Peróxido de Hidrogênio , Masculino , Ratos Sprague-Dawley , Heme Oxigenase-1/metabolismo , CamundongosRESUMO
Vascular defects caused by trauma or vascular diseases can significantly impact normal blood circulation, resulting in serious health complications. Vascular grafts have evolved as a popular approach for vascular reconstruction with promising outcomes. However, four of the greatest challenges for successful application of small-diameter vascular grafts are (1) postoperative anti-infection, (2) preventing thrombosis formation, (3) utilizing the inflammatory response to the graft to induce tissue regeneration and repair, and (4) noninvasive monitoring of the scaffold and integration. The present study demonstrated a basic fibroblast growth factor (bFGF) and oleic acid dispersed Ag@Fe3O4 core-shell nanowires (OA-Ag@Fe3O4 CSNWs) codecorated poly(lactic acid) (PLA)/gelatin (Gel) multifunctional electrospun vascular grafts (bAPG). The Ag@Fe3O4 CSNWs have sustained Ag+ release and exceptional photothermal capabilities to effectively suppress bacterial infections both in vitro and in vivo, noninvasive magnetic resonance imaging (MRI) modality to monitor the position of the graft, and antiplatelet adhesion properties to promise long-term patency. The gradually released bFGF from the bAPG scaffold promotes the M2 macrophage polarization and enhances the recruitment of macrophages, endothelial cells (ECs) and fibroblast cells. This significant regulation of diverse cell behavior has been proven to be beneficial to vascular repair and regeneration both in vitro and in vivo. Therefore, this study supplies a method to prepare multifunctional vascular-repair materials and is expected to represent a significant guidance and reference to the development of biomaterials for vascular tissue engineering.
Assuntos
Fator 2 de Crescimento de Fibroblastos , Gelatina , Nanofibras , Nanofios , Poliésteres , Prata , Alicerces Teciduais , Poliésteres/química , Gelatina/química , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/farmacologia , Animais , Prata/química , Nanofibras/química , Nanofios/química , Alicerces Teciduais/química , Humanos , Prótese Vascular , Camundongos , Células Endoteliais da Veia Umbilical HumanaRESUMO
Understanding the factors which control endothelial cell (EC) function and angiogenesis is crucial for developing the horse as a disease model, but equine ECs remain poorly studied. In this study, we have optimised methods for the isolation and culture of equine aortic endothelial cells (EAoECs) and characterised their angiogenic functions in vitro. Mechanical dissociation, followed by magnetic purification using an anti-VE-cadherin antibody, resulted in EC-enriched cultures suitable for further study. Fibroblast growth factor 2 (FGF2) increased the EAoEC proliferation rate and stimulated scratch wound closure and tube formation by EAoECs on the extracellular matrix. Pharmacological inhibitors of FGF receptor 1 (FGFR1) (SU5402) or mitogen-activated protein kinase (MEK) (PD184352) blocked FGF2-induced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and functional responses, suggesting that these are dependent on FGFR1/MEK-ERK signalling. In marked contrast, vascular endothelial growth factor-A (VEGF-A) had no effect on EAoEC proliferation, migration, or tubulogenesis and did not promote ERK1/2 phosphorylation, indicating a lack of sensitivity to this classical pro-angiogenic growth factor. Gene expression analysis showed that unlike human ECs, FGFR1 is expressed by EAoECs at a much higher level than both VEGF receptor (VEGFR)1 and VEGFR2. These results suggest a predominant role for FGF2 versus VEGF-A in controlling the angiogenic functions of equine ECs. Collectively, our novel data provide a sound basis for studying angiogenic processes in horses and lay the foundations for comparative studies of EC biology in horses versus humans.
Assuntos
Proliferação de Células , Células Endoteliais , Fator 2 de Crescimento de Fibroblastos , Neovascularização Fisiológica , Fator A de Crescimento do Endotélio Vascular , Animais , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Cavalos , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Proliferação de Células/efeitos dos fármacos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosforilação/efeitos dos fármacosRESUMO
OBJECTIVE: This study examined how range concentrations of Fibroblast Growth Factor-2 (FGF-2) influence the differentiation and activity of human-derived periodontal ligament (hPDLSCs) and alveolar bone-derived stem cells (haBMSCs). DESIGN: hPDLSCs and haBMSCs were cultured with varying concentrations of FGF-2 (0, 1, 2.5, 5, 10, 20 ng/mL) and monitored for osteogenic differentiation through alkaline phosphatase (ALP) activity and quantification of gene expression (qRT-PCR) for osteogenesis markers. Additionally, alizarin red staining and a hydroxyproline colorimetric assay evaluated and quantified osteogenic matrix mineralization and collagen deposition. Statistical analyses were performed using one-way ANOVA or two-way ANOVA for multiple comparisons between groups. RESULTS: At low FGF-2 concentrations, hPDLSCs differentiated toward an osteogenic lineage, whereas higher concentrations of FGF-2 inhibited osteogenesis and promoted fibroblastic differentiation. The effect of FGF-2 at the lowest concentration tested (1 ng/mL) led to significantly higher ALP activity than osteogenically induced positive controls at early time points and equivalent RUNX2 expression at early and later time points. FGF-2 supplementation of haBMSC cultures was sufficient, at all concentrations, to increase ALP activity at an earlier time point. Mineralization of haBMSC cultures increased significantly within 5-20 ng/mL FGF-2 concentrations under basal growth media conditions (α-minimal essential medium supplemented with 15 % fetal bovine serum and 1 % penicillin/streptomycin). CONCLUSIONS: FGF-2 has a dual capacity in promoting osteogenic and fibroblastic differentiation within hPDLSCs contingent upon the dosage and timing of administration, alongside supporting osteogenic differentiation in haBMSCs. These findings underscore the need for precision growth factors dosing when considering the design of biomaterials for periodontal regeneration.
Assuntos
Fator 2 de Crescimento de Fibroblastos , Ligamento Periodontal , Humanos , Fosfatase Alcalina/metabolismo , Processo Alveolar/citologia , Processo Alveolar/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Ligamento Periodontal/citologia , Ligamento Periodontal/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Células-Tronco/efeitos dos fármacosRESUMO
This work employs nitrogen plasma immersion ion implantation (PIII) to modify electrospinning polylactic acid membranes and immobilizes basic fibroblast growth factors (bFGF) by forming crosslinking bonds. The study investigates the modified membranes' surface characteristics and the stimulatory effects of crosslinked bFGF polylactic acid membranes on osteoblast and fibroblast proliferation. The PIII process occurs under low vacuum conditions and is controlled by processing time and power pulse width. The experimental results indicate that, within a 400-second N2-PIII treatment, the spun fibers remain undamaged, demonstrating an increase in hydrophilicity (from 117° to 38°/36°) and nitrogen content (from 0% to 7.54%/8.05%). X-ray photoelectron spectroscopy analysis suggests the formation of a C-N-C=O crosslinked bond. Cell culture and activity assessments indicate that the PIII-treated and crosslinked bFGF film exhibits significantly higher cell growth activity (p < 0.05) than the untreated group. These intergroup differences are attributed to the surface crosslinking bond content. In osteogenic induction, the results for each day show that the treated group performs better. However, the intergroup disparities within the crosslinked bFGF group disappear with prolonged culture time due to the rapid osteogenesis prompted by bFGF. The findings suggest that PIII treatment of electrospinning polylactic acid membranes holds promise in promoting osteogenesis in bone tissue scaffolds.
Assuntos
Materiais Biocompatíveis , Diferenciação Celular , Proliferação de Células , Nanofibras , Osteoblastos , Nanofibras/química , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Materiais Biocompatíveis/farmacologia , Materiais Biocompatíveis/química , Animais , Poliésteres/química , Poliésteres/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/química , Gases em Plasma/farmacologia , Camundongos , Osteogênese/efeitos dos fármacos , Ácido Láctico/química , Ácido Láctico/farmacologia , Espectroscopia FotoeletrônicaRESUMO
Protein materials are versatile tools in diverse biomedical fields. Among them, artificial secretory granules (SGs), mimicking those from the endocrine system, act as mechanically stable reservoirs for the sustained release of proteins as oligomeric functional nanoparticles. Only validated in oncology, the physicochemical properties of SGs, along with their combined drug-releasing and scaffolding abilities, make them suitable as smart topographies in regenerative medicine for the prolonged delivery of growth factors (GFs). Thus, considering the need for novel, safe, and cost-effective materials to present GFs, in this study, we aimed to biofabricate a protein platform combining both endocrine-like and extracellular matrix fibronectin-derived (ECM-FN) systems. This approach is based on the sustained delivery of a nanostructured histidine-tagged version of human fibroblast growth factor 2. The GF is presented onto polymeric surfaces, interacting with FN to spontaneously generate nanonetworks that absorb and present the GF in the solid state, to modulate mesenchymal stromal cell (MSC) behavior. The results show that SGs-based topographies trigger high rates of MSCs proliferation while preventing differentiation. While this could be useful in cell therapy manufacture demanding large numbers of unspecialized MSCs, it fully validates the hybrid platform as a convenient setup for the design of biologically active hybrid surfaces and in tissue engineering for the controlled manipulation of mammalian cell growth.
Assuntos
Matriz Extracelular , Fibronectinas , Células-Tronco Mesenquimais , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Fibronectinas/química , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/farmacologia , Proliferação de Células/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Nanoestruturas/químicaRESUMO
Synthetic vascular grafts are used to bypass significant arterial blockage when native blood vessels are unsuitable, yet their propensity to fail due to poor blood compatibility and progressive graft stenosis remains an intractable challenge. Perlecan is the major heparan sulfate (HS) proteoglycan in the blood vessel wall with an inherent ability to regulate vascular cell activities associated with these major graft failure modes. Here the ability of the engineered form of perlecan domain V (rDV) to bind angiogenic growth factors is tuned and endothelial cell proliferation via the composition of its glycosaminoglycan (GAG) chain is supported. It is shown that the HS on rDV supports angiogenic growth factor signaling, including fibroblast growth factor (FGF) 2 and vascular endothelial growth factor (VEGF)165, while both HS and chondroitin sulfate on rDV are involved in VEGF189 signaling. It is also shown that physisorption of rDV on emerging electrospun silk fibroin vascular grafts promotes endothelialization and patency in a murine arterial interposition model, compared to the silk grafts alone. Together, this study demonstrates the potential of rDV as a tunable, angiogenic biomaterial coating that both potentiates growth factors and regulates endothelial cells.