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1.
Bone Joint J ; 102-B(8): 1095-1106, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32731821

RESUMO

AIMS: Achilles tendon injuries are a frequent problem in orthopaedic surgery due to their limited healing capacity and the controversy surrounding surgical treatment. In recent years, tissue engineering research has focused on the development of biomaterials to improve this healing process. The aim of this study was to analyze the effect of tendon augmentation with a nanostructured fibrin-agarose hydrogel (NFAH) or genipin cross-linked nanostructured fibrin-agarose hydrogel (GP-NFAH), on the healing process of the Achilles tendon in rats. METHODS: NFAH, GP-NFAH, and MatriDerm (control) scaffolds were generated (five in each group). A biomechanical and cell-biomaterial-interaction characterization of these biomaterials was then performed: Live/Dead Cell Viability Assay, water-soluble tetrazolium salt-1 (WST-1) assay, and DNA-released after 48 hours. Additionally, a complete section of the left Achilles tendon was made in 24 Wistar rats. Animals were separated into four treatment groups (six in each group): direct repair (Control), tendon repair with MatriDerm, or NFAH, or GP-NFAH. Animals were euthanized for further histological analyses after four or eight weeks post-surgery. The Achilles tendons were harvested and a histopathological analysis was performed. RESULTS: Tensile test revealed that NFAH and GP-NFAH had significantly higher overall biomechanical properties compared with MatriDerm. Moreover, biological studies confirmed a high cell viability in all biomaterials, especially in NFAH. In addition, in vivo evaluation of repaired tendons using biomaterials (NFAH, GP-NFAH, and MatriDerm) resulted in better organization of the collagen fibres and cell alignment without clinical complications than direct repair, with a better histological score in GP-NFAH. CONCLUSION: In this animal model we demonstrated that NFAH and GP-NFAH had the potential to improve tendon healing following a surgical repair. However, future studies are needed to determine the clinical usefulness of these engineered strategies. Cite this article: Bone Joint J 2020;102-B(8):1095-1106.


Assuntos
Tendão do Calcâneo/cirurgia , Microambiente Celular/efeitos dos fármacos , Colágeno/uso terapêutico , Elastina/uso terapêutico , Regeneração/efeitos dos fármacos , Traumatismos dos Tendões/cirurgia , Tendão do Calcâneo/lesões , Animais , Materiais Biocompatíveis/farmacologia , Modelos Animais de Doenças , Fibrina/farmacologia , Hidrogéis/farmacologia , Masculino , Nanoestruturas , Distribuição Aleatória , Ratos , Ratos Wistar , Tendões/fisiologia , Engenharia Tecidual/métodos , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologia
2.
Sci Rep ; 10(1): 1850, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-32024893

RESUMO

Platelet-rich fibrin (PRF) provides a scaffold for cell migration and growth factors for promoting wound healing and tissue regeneration. Here, we report using PRF in periodontal healing after open flap debridement (OFD) in canine periodontitis. A split-mouth design was performed in twenty dogs. Forty periodontitis surgical sites were randomly categorized into 2 groups; OFD alone and OFD with PRF treatment. Clinical parameters of periodontal pocket depth, gingival index, and the cemento-enamel junction-alveolar bone levels/root length ratio were improved in the OFD + PRF group. The OFD + PRF group also demonstrated a dramatically decreased inflammatory score compared with the OFD group. Collagen accumulation was improved in the OFD + PRF group at later time points compared with baseline. PRF application also significantly reduced inflammatory cytokine expression (TNFA and IL1B), and promoted the expression of collagen production-related genes (COL1A1, COL3A1, and TIMP1) and growth factors (PDGFB, TGFB1, and VEGFA). These findings suggest that PRF combined with OFD provides a new strategy to enhance the overall improvement of canine periodontitis treatment outcomes, especially in terms of inflammation and soft tissue healing. Therefore, PRF use in treating periodontitis could play an important role as a regenerative material to improve canine periodontitis treatment.


Assuntos
Periodontite Crônica/metabolismo , Fibrina/farmacologia , Bolsa Periodontal/tratamento farmacológico , Bolsa Periodontal/metabolismo , Fibrina Rica em Plaquetas/metabolismo , Regeneração/efeitos dos fármacos , Regeneração/fisiologia , Perda do Osso Alveolar/tratamento farmacológico , Perda do Osso Alveolar/metabolismo , Animais , Citocinas/metabolismo , Desbridamento/métodos , Cães , Genes Reguladores/genética , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Índice Periodontal , Retalhos Cirúrgicos/fisiologia , Resultado do Tratamento , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologia
3.
J Vis Exp ; (166)2020 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-33427234

RESUMO

Drosophila is an important model system to study a vast range of biological questions. Various organs and tissues from different developmental stages of the fly such as imaginal discs, the larval brain or egg chambers of adult females or the adult intestine can be extracted and kept in culture for imaging with time-lapse microscopy, providing valuable insights into cell and developmental biology. Here, we describe in detail our current protocol for the dissection of Drosophila larval brains, and then present our current approach for immobilizing and orienting larval brains and other tissues on a glass coverslip using Fibrin clots. This immobilization method only requires the addition of Fibrinogen and Thrombin to the culture medium. It is suitable for high-resolution time lapse imaging on inverted microscopes of multiple samples in the same culture dish, minimizes the lateral drifting frequently caused by movements of the microscope stage in multi-point visiting microscopy and allows for the addition and removal of reagents during the course of imaging. We also present custom-made macros that we routinely use to correct for drifting and to extract and process specific quantitative information from time-lapse analysis.


Assuntos
Coagulação Sanguínea , Drosophila melanogaster/fisiologia , Fibrina/farmacologia , Imageamento Tridimensional , Trifosfato de Adenosina/análogos & derivados , Animais , Encéfalo/anatomia & histologia , Meios de Cultura , Dissecação , Feminino , Discos Imaginais , Imobilização , Larva/citologia , Óvulo/efeitos dos fármacos , Óvulo/fisiologia , Processamento de Sinais Assistido por Computador
4.
Toxicol Appl Pharmacol ; 385: 114811, 2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31705944

RESUMO

In vivo local antitumor activity of fibrin gels (FBGs) loaded with the poly-cyclodextrin oCD-NH2/Dox, compared to free Dox, was evaluated in two mouse orthotopic neuroblastoma (NB) models, after positioning of the releasing devices in the visceral space. FBGs were prepared at the fibrinogen (FG) concentrations of 22 and 40 mg/ml clotted in the presence of 0.81 mM/mg FG Ca2+ and 1.32 U/mg FG thrombin. Our results indicate that FBGs loaded with oCD-NH2/Dox and applied as neoadjuvant loco-regional treatment, show an antitumor activity significantly greater than that displayed by the same FBGs loaded with identical dose of Dox or after free Dox administered intra venous (iv). In particular, FBGs prepared at 40 mg/ml showed a slightly lower antitumor activity, although after their positioning we observed a significant initial reduction of tumor burden lasting for several days after gel implantation. FBGs at 22 mg/ml loaded with oCD-NH2/Dox and applied after tumor removal (adjuvant treatment model) showed a significantly better antitumor activity than the iv administration of free Dox, with 90% tumor regrowth reduction compared to untreated controls. In all cases the weight loss post-treatment was limited after gel application, although in the adjuvant treatment the loss of body weight lasted longer than in the other treatment modality. In accordance with our recent published data on the low local toxic effects of FBGs, the present findings also underline an increase of the therapeutic index of Dox when locally administered through FBGs loaded with the oCD-NH2/Dox complex.


Assuntos
Celulose/química , Ciclodextrinas/química , Doxorrubicina/administração & dosagem , Fibrina/administração & dosagem , Neuroblastoma/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Feminino , Fibrina/farmacologia , Fibrina/toxicidade , Géis , Humanos , Camundongos , Terapia Neoadjuvante , Neuroblastoma/patologia
5.
Eur Cell Mater ; 38: 201-214, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31682278

RESUMO

Dental implants are the usual therapy of choice in the dental clinic to replace a loss of natural teeth. Over recent decades there has been an important progress in the design and manufacturing of titanium implant surfaces with the goal of improving their osteointegration. In the present work, the aim was to evaluate the usefulness of hDPSCs (human dental pulp stem cells), in combination with autologous plasma components, for in vitro bone generation on biomimetic titanium dental implant materials. In this context, the combination of hDPSCs stimulated by PRGF or PRF and cultured on standard Ti6A14V and biomimetic BAS™ (Avinent Implant System) titanium surfaces were studied in order to evaluate possible enhancements in the osteoblastic differentiation process out of human mesenchymal cells, as well as bone matrix secretion on the implant surface. The results obtained in this in vitro model of osteogenesis suggested a combination of biomimetic rough titanium surfaces, such as BAS™, with autologous plasma-derived fibrin-clot membranes such as PRF and/or insoluble PRGF formulations, but not with an addition of water-soluble supplements of plasma-derived growth factors, to maximise osteoblastic cell differentiation, bone generation, anchorage and osteointegration of titanium-made dental implants.


Assuntos
Materiais Biomiméticos/química , Adesão Celular , Polpa Dentária/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osseointegração , Osteogênese , Plasma Rico em Plaquetas/química , Adulto , Materiais Biomiméticos/farmacologia , Células Cultivadas , Implantes Dentários , Fibrina/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Titânio/química
6.
Mater Sci Eng C Mater Biol Appl ; 104: 109847, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31500049

RESUMO

Hydrogel matrices with angiogenic properties are much desirable for therapeutic vascularization strategies, namely to provide vascular supply to ischemic areas, transplanted cells, or bioengineered tissues. Here we report the pro-angiogenic effect of fibrin (Fb) functionalization with the T1 sequence from the angiogenic inducer CCN1, forseeing its use in the injured brain and spinal cord. Fibrin functionalization with 40 µM of T1 peptide effectively improved cellular sprouting of human brain microvascular endothelial cells (hCMEC/D3) in the absence of vascular endothelial growth factor (VEGF), without impacting the viscoelastic properties of Fb, cell viability, or proliferation. The pro-angiogenic effect of immobilized T1 was potentiated in the presence of VEGF and partially mediated through α6ß1 integrin. The tethering of T1 also enhanced sprouting of human cord blood-derived outgrowth endothelial cells (OEC). Still, to elicit such effect, a higher input T1 concentration was required (60 µM), in line with the lower protein levels of α6 and ß1 integrin subunits found in OEC comparing to hCMEC/D3, prior to embedment in Fb gel. Finally, the ability of T1-functionalized Fb in inducing cappilary invasion in vivo was assessed using the CAM assay, which evidenced a significant increase in the number of newly formed vessels at sites of implantation of T1-functionalized Fb, in the absence of soluble angiogenic factors. Overall these results demonstrate the potential of T1 peptide-presenting gels for use in therapeutic vascularization approaches. Considering T1 neurite-extension promoting capability and pro-angiogenic properties, T1-functionalized Fb hydrogels are particularly promising for application in the injured central nervous system.


Assuntos
Proteína Rica em Cisteína 61/química , Proteína Rica em Cisteína 61/farmacologia , Fibrina/farmacologia , Hidrogéis/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Galinhas , Membrana Corioalantoide/efeitos dos fármacos , Elasticidade , Células Endoteliais/efeitos dos fármacos , Humanos , Viscosidade
7.
Mater Sci Eng C Mater Biol Appl ; 104: 109931, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31499978

RESUMO

Fibrin gels are of interest as biomaterials for regenerative medicine but present poor mechanical properties, undergo fast degradation and strongly contract in presence of cells. To face these drawbacks, a fibrin network can be associated with another polymer network, in an Interpenetrating Polymer Network (IPN) architecture. In this study, we report the properties of an IPN comprising a fibrin (Fb) network and a silk fibroin (SF) network. This IPN is synthesized through the action of 2 enzymes, each one being specific of one protein gelation, i.e. thrombin (Tb) for Fb gelation, and horseradish peroxidase (HRP) for SF gelation. The effective formation of both Fb and SF networks in an IPN architecture was first verified at qualitative and quantitative levels. The resulting IPN was easily manipulable, displayed high viscoelastic properties and showed homogeneous macro- and micro-structure. Then the degradability of the IPN by two proteases, thermolysin (TL) and trypsin (TRY), obeying different mechanisms was presented. Finally, two-dimensional culture of human fibroblasts on the IPN surface induced little material contraction, while fibroblasts showed healthy morphology, displayed high viability and produced mature extracellular matrix (ECM) proteins. Taken together, the results suggest that this new IPN have a strong potential for tissue engineering and regenerative medicine.


Assuntos
Fibrina/farmacologia , Fibroínas/farmacologia , Peroxidase do Rábano Silvestre/metabolismo , Polímeros/farmacologia , Trombina/metabolismo , Proliferação de Células , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/biossíntese , Fibronectinas/biossíntese , Humanos , Recém-Nascido , Masculino
8.
Int J Nanomedicine ; 14: 5033-5050, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31371945

RESUMO

Background: Repairs to deep skin wounds continue to be a difficult issue in clinical practice. A promising approach is to fabricate full-thickness skin substitutes with functions closely similar to those of the natural tissue. For many years, a three-dimensional (3D) collagen hydrogel has been considered to provide a physiological 3D environment for co-cultivation of skin fibroblasts and keratinocytes. This collagen hydrogel is frequently used for fabricating tissue-engineered skin analogues with fibroblasts embedded inside the hydrogel and keratinocytes cultivated on its surface. Despite its unique biological properties, the collagen hydrogel has insufficient stiffness, with a tendency to collapse under the traction forces generated by the embedded cells. Methods: The aim of our study was to develop a two-layer skin construct consisting of a collagen hydrogel reinforced by a nanofibrous poly-L-lactide (PLLA) membrane pre-seeded with fibroblasts. The attractiveness of the membrane for dermal fibroblasts was enhanced by coating it with a thin nanofibrous fibrin mesh. Results: The fibrin mesh promoted the adhesion, proliferation and migration of the fibroblasts upwards into the collagen hydrogel. Moreover, the fibroblasts spontaneously migrating into the collagen hydrogel showed a lower tendency to contract and shrink the hydrogel by their traction forces. The surface of the collagen was seeded with human dermal keratinocytes. The keratinocytes were able to form a basal layer of highly mitotically-active cells, and a suprabasal layer. Conclusion: The two-layer skin construct based on collagen hydrogel with spontaneously immigrated fibroblasts and reinforced by a fibrin-coated nanofibrous membrane seems to be promising for the construction of full-thickness skin substitute.


Assuntos
Colágeno/farmacologia , Fibrina/farmacologia , Hidrogéis/farmacologia , Membranas Artificiais , Nanofibras/química , Poliésteres/farmacologia , Pele Artificial , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Derme/citologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Recém-Nascido , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ratos
9.
Tissue Eng Part C Methods ; 25(9): 523-531, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31418322

RESUMO

IMPACT STATEMENT: Noninvasive imaging techniques provide insight into physiology that is complementary to tissue morphology obtained by invasive histology. Optical imaging techniques, such as laser speckle contrast analysis, are used in vivo to longitudinally evaluate vascularization. Despite their high spatial resolution, these techniques have a limited imaging depth. In this study, we demonstrate how a dual LED-based photoacoustic (PA) and ultrasound system can delineate changes in perfusion at depth within scaffolds containing basic fibroblast growth factor. Perfusion changes detected by PA corroborated with vessel density. PA imaging could be a noninvasive and sensitive method for evaluating vascularization at depth in larger constructs.


Assuntos
Fibrina , Fator 2 de Crescimento de Fibroblastos , Neovascularização Fisiológica/efeitos dos fármacos , Técnicas Fotoacústicas , Animais , Implantes de Medicamento/farmacocinética , Implantes de Medicamento/farmacologia , Feminino , Fibrina/farmacocinética , Fibrina/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacocinética , Fator 2 de Crescimento de Fibroblastos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C
10.
Indian J Med Res ; 149(5): 641-649, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31417032

RESUMO

Background & objectives: Seeding density is one of the major parameters affecting the quality of tissue-engineered cartilage. The objective of this study was to evaluate different seeding densities of osteoarthritis chondrocytes (OACs) to obtain the highest quality cartilage. Methods: The OACs were expanded from passage 0 (P0) to P3, and cells in each passage were analyzed for gross morphology, growth rate, RNA expression and immunochemistry (IHC). The harvested OACs were assigned into two groups: low (1×10[7] cells/ml) and high (3×10[7] cells/ml) cell density. Three-dimensional (3D) constructs for each group were created using polymerised fibrin and cultured for 7, 14 and 21 days in vitro using chondrocyte growth medium. OAC constructs were analyzed with gross assessments and microscopic evaluation using standard histology, IHC and immunofluorescence staining, in addition to gene expression and biochemical analyses to evaluate tissue development. Results: Constructs with a high seeding density of 3×10[7] cells/ml were associated with better quality cartilage-like tissue than those seeded with 1×10[7] cells/ml based on overall tissue formation, cell association and extracellular matrix distribution. The chondrogenic properties of the constructs were further confirmed by the expression of genes encoding aggrecan core protein and collagen type II. Interpretation & conclusions: Our results confirmed that cell density was a significant factor affecting cell behaviour and aggregate production, and this was important for establishing good quality cartilage.


Assuntos
Cartilagem/crescimento & desenvolvimento , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Osteoartrite/terapia , Cartilagem/efeitos dos fármacos , Cartilagem Articular , Técnicas de Cultura de Células/métodos , Condrócitos/metabolismo , Condrogênese/efeitos dos fármacos , Fibrina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Osteoartrite/patologia , Osteogênese/efeitos dos fármacos , RNA/genética
11.
Med Sci Monit ; 25: 4384-4389, 2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-31188801

RESUMO

BACKGROUND The restoration of damaged periodontium, especially one-wall intrabony defects, is a major challenge for clinicians. Concentrated growth factors (CGF) are a 100% autologous fibrin with multiple concentrated growth factors. The rigid fibrin structure of CGF makes it possible to preserve or reconstruct the initial bone volume. The aim of this study was to evaluate the clinical healing patterns after surgical application of CGF with and without a Bio-Oss graft in one-wall infrabony defects. MATERIAL AND METHODS We randomly divided 120 one-wall intrabony defects in 54 patients into 4 groups: flap surgery alone (Group 1), flap surgery with autologous CGF (Group 2), flap surgery with Bio-Oss (Group 3), and flap surgery with CGF+Bio-Oss (Group 4). Clinical parameters such as probing depth (PD) and clinical attachment level (CAL) change were recorded at baseline and at 6 and 12 months postoperatively. RESULTS At 12 months postoperatively, Group 2 showed significant improvement in clinical parameters over Group 1 (P<0.05) and the results were significantly greater in Groups 3 and 4 compared to the other groups (P<0.05). Although no significant difference was noted between Groups 3 and 4 in clinical parameters (P>0.05) compared to Group 3, the mean change of CAL at 6-12 months in Group 4 was not significant (P>0.05). CONCLUSIONS CGF reduced periodontal intrabony defects depth and, when mixed with Bio-Oss, CGF showed better results in the early period and the effect was more stable.


Assuntos
Substitutos Ósseos/farmacologia , Periodontite Crônica/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Adulto , Perda do Osso Alveolar/tratamento farmacológico , Regeneração Óssea/efeitos dos fármacos , China , Feminino , Fibrina/farmacologia , Seguimentos , Humanos , Masculino , Doenças Mandibulares/tratamento farmacológico , Pessoa de Meia-Idade , Minerais/farmacologia , Índice Periodontal , Ligamento Periodontal , Cicatrização/efeitos dos fármacos
12.
Sci Rep ; 9(1): 8188, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160643

RESUMO

Various studies have described the biological properties of the Leucocyte- and Platelet Rich Fibrin (L-PRF) such as the antimicrobial effect against wound bacteria, but less is known about the effect against periodontal pathogens. The aim of this study was to evaluate the antibacterial properties of the L-PRF membrane and L-PRF exudate against the main periopathogens cultured on agar plates and in planktonic solution. This study demonstrated the antibacterial effect of the L-PRF membrane against P. intermedia, F. nucleatum, and A. actinomycetemcomitans, but especially against P. gingivalis. The L-PRF exudate also showed a strong inhibition against P. gingivalis on agar plates. No inhibition could be observed for the other bacterial strains. Moreover, L-PRF exudate decreased the number of viable P.gingivalis in a planktonic solution in a dose-dependent way. However, A. actinomycetemcomitans showed an increased growth in planktonic solution when in contact with the L-PRF exudate.


Assuntos
Antibacterianos/farmacologia , Leucócitos/citologia , Periodonto/microbiologia , Fibrina Rica em Plaquetas , Adulto , Ágar , Aggregatibacter actinomycetemcomitans , Clorexidina/farmacologia , Feminino , Fibrina/farmacologia , Fusobacterium nucleatum , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Doenças Periodontais/microbiologia , Periodonto/efeitos dos fármacos , Reação em Cadeia da Polimerase , Porphyromonas gingivalis , Prevotella intermedia
13.
Cell Tissue Res ; 378(1): 127-141, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31049685

RESUMO

Differentiation of mesenchymal stem cells (MSCs) into cardiomyocytes is a complex phenomenon, and attempts to find an effective inducing agent are still ongoing. We studied the effect of fibrin scaffold and sodium valproate (VPA, as a histone deacetylase inhibitor) on the differentiation of human adipose-derived stem cells (hADSCs) into cardiomyocyte-like cells. The cells were cultured in culture flask (2D) and in fibrin scaffold (3D), fabricated of human plasma fibrinogen, with and without VPA (1 mM). QRT-PCR, Western blot, and immunochemistry assays were used to evaluate the expression of cardiac markers at gene and protein levels. High levels of CD44, CD90, CD73, and CD105 were expressed on the surface of hADSCs. Treated encapsulated hADSCs (3D) presented significantly higher mRNA expression of HAND1 (1.54-fold), HAND2 (1.59-fold), cTnI (1.76-fold), MLC2v (1.4-fold), Cx43 (1.38-fold), ßMHC (1.34-fold), GATA4 (1.48-fold), and NKX2.5 (1.66-fold) in comparison to 2D conditions at four weeks after induction. The protein expressions of NKX2.5 (0.78 vs 0.65), cTnI (1.04 vs 0.81), and Cx43 (1.11 vs 1.08) were observed in the differentiated cells both in 3D and 2D groups, while control cells were absolutely negative for these proteins. The frequency of cTnI and Cx43-positive cells was significantly higher in 3D (24.2 ± 15 and 12 ± 3%) than 2D conditions (19.8 ± 3 and 10 ± 2%). Overall, the results showed that VPA can increase cardiomyogenesis in hADSCs and that fibrin scaffold enhances the inductive effect of VPA. Results of this study may improve cell-based protocols for implementation of more successful cardiac repair strategies.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Fibrina/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Células-Tronco Mesenquimais/citologia , Ácido Valproico/farmacologia , Células Cultivadas , Humanos , Tecidos Suporte
14.
PLoS One ; 14(5): e0217601, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31120999

RESUMO

A cell therapy product of transforming growth factor (TGF)-ß1-transduced chondrocytes has been commercialized to treat osteoarthritis of the knee via intra-articular injection. The need for arthroscopic application of the cells to simultaneously treat intra-articular pathologies of knee osteoarthritis is increasingly urgent. The purpose of this study was to optimize TGF-ß1-transduced chondrocytes for arthroscopic application. The optimal composition of chondrocytes and thrombin was initially determined by measuring the consolidation time of a diverse ratio of chondrocytes and thrombin mixed with 1 ml of fibrinogen. The consolidation time of the diverse ratio of fibrinogen and atelocollagen mixed with the determined optimal ratio of chondrocytes and thrombin was evaluated. The mixture of the determined optimal ratio of TGF-ß1-transduced chondrocytes, atelocollagen, fibrinogen, and thrombin was applied to the cartilage defect of the 3D printed knee joint model arthroscopically. The status of the mixture in the defect was then evaluated. Chondrogenic activities of TGF-ß1-transduced chondrocytes mixed with atelocollagen were evaluated. The determined ratio of TGF-ß1-transduced chondrocytes to thrombin was 8:2 and that of fibrin to atelocollagen was also 8:2. Excellent maintenance of conformation of the mixture of TGF-ß1-transduced chondrocytes, atelocollagen, fibrinogen, and thrombin in the cartilage defect of the 3D printed knee joint model was observed arthroscopically. Increased chondrogenic activities were observed in the group of TGF-ß1-transduced chondrocytes mixed with atelocollagen. TGF-ß1-transduced chondrocytes can be applied arthroscopically to treat cartilage defects of the knee at an optimized mixing ratio of atelocollagen, fibrinogen, and thrombin.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Osteoartrite/tratamento farmacológico , Fator de Crescimento Transformador beta1/genética , Artroscopia , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Cartilagem/patologia , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Colágeno/química , Colágeno/farmacologia , Fibrina/química , Fibrina/farmacologia , Fibrinogênio/química , Fibrinogênio/farmacologia , Humanos , Injeções Intra-Articulares , Articulação do Joelho/efeitos dos fármacos , Articulação do Joelho/patologia , Osteoartrite/genética , Osteoartrite/patologia , Impressão Tridimensional , Regeneração/efeitos dos fármacos , Regeneração/genética , Trombina/química , Trombina/farmacologia
15.
J Tissue Eng Regen Med ; 13(8): 1362-1374, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31067012

RESUMO

Cultured epithelial autografts (CEAs) represent a life-saving surgical technique for full-thickness skin burns covering more than 60% total body surface area. However, CEAs present numerous drawbacks leading to heavy cosmetic and functional sequelae. In our previous study, we showed that human plasma-based fibrin matrices (hPBM) could improve the reparative potential of CEAs. Therefore, in the present work, we sought to investigate the role of hPBM compared with fibrin from purified fibrinogen (FPF) or plastic support on epidermal substitute formation and engraftment. The use of hPBM for epidermal substitute culture improved keratinocyte migration, proliferation, and epidermal substitute organization to a better extent than FPF in vitro. Both fibrin matrices favored greater dermal-epidermal junction protein deposition and prevented their degradation. Keratinocyte differentiation was also decreased using both fibrin matrices. Basement membrane protein deposition was mainly influenced by matrix whereas growth factors released from fibrin especially by hPBM were shown to enhance in vitro keratinocyte migration, proliferation, and epidermal substitute organization. Ultimately, epidermal substitutes grown on hPBM displayed better engraftment rates than those cultured on FPF or on plastic support in a NOD-SCID model of acute wound with the formation of a functional dermal-epidermal junction. Together, these results show the positive impact of fibrin matrices and their released growth factor on epidermal substitute phenotype and grafting efficiency. Fibrin matrices, and especially hPBM, may therefore be of interest to favor the treatment of full-thickness burn patients.


Assuntos
Epiderme/efeitos dos fármacos , Fibrina/farmacologia , Transplante de Pele , Pele Artificial , Doença Aguda , Animais , Membrana Basal/efeitos dos fármacos , Membrana Basal/metabolismo , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Epiderme/ultraestrutura , Feminino , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Fenótipo , Engenharia Tecidual
16.
ACS Appl Mater Interfaces ; 11(20): 18123-18132, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31026135

RESUMO

In situ injectable hydrogels hold great potential for in vivo applications such as drug delivery and regenerative medicine. However, it is challenging to ensure stable sequestration and sustained release of loaded biomolecules in these hydrogels. As aptamers have high binding affinities and specificities against target biomolecules, we studied the capability of aptamers in functionalizing in situ injectable fibrin (Fn) hydrogels for in vivo delivery of two growth factors including vascular endothelial growth factor (VEGF) and platelet-derived growth factor-BB (PDGF-BB). The results show that aptamer-functionalized fibrinogen (Fg) could form in situ injectable Fn hydrogels with porous structures. The aptamer-functionalized Fn hydrogels could sequester more VEGF and PDGF-BB than the native Fn and release these growth factors in a sustained manner with high bioactivity. After the aptamer-functionalized Fn hydrogels were subcutaneously injected into mice, the codelivery of VEGF and PDGF-BB could promote the growth of mature blood vessels. Therefore, this study has successfully demonstrated that aptamer-functionalized in situ injectable hydrogels hold great potential for in vivo codelivery of multiple growth factors and promotion of angiogenesis .


Assuntos
Aptâmeros de Nucleotídeos , Becaplermina , Fibrina , Hidrogéis , Neovascularização Fisiológica/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular , Animais , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/farmacologia , Becaplermina/química , Becaplermina/farmacologia , Feminino , Fibrina/química , Fibrina/farmacologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Hidrogéis/química , Hidrogéis/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/farmacologia
17.
Acta Biomater ; 95: 348-356, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30831326

RESUMO

Stem cell encapsulation in hydrogels has been widely employed in tissue engineering, regenerative medicine, organ-on-a-chip devices and gene delivery; however, fabrication of native-like bone tissue using such a strategy has been a challenge, particularly in vitro, due to the limited cell loading densities resulting in weaker cell-cell interactions and lesser extra-cellular matrix deposition. In particular, scalable bone tissue constructs require vascular network to provide enough oxygen and nutrient supplies to encapsulated cells. To enhance stem cell function and generate pre-vascularized network, we here employed collagen/fibrin hydrogel as an encapsulation matrix for the incorporation of human mesenchymal stem cell/human umbilical vein endothelial cell (MSC/HUVEC) spheroids, and investigated their cellular behavior (including cell viability, morphology, proliferation, and gene expression profile) and compared to that of cell suspension- or MSC spheroids-laden hydrogels. MSC/HUVEC spheroids encapsulated in collagen/fibrin hydrogel showed better cell spreading and proliferation, and up-regulated osteogenic differentiation, and demonstrated pre-vascular network formation. Overall, MSC/HUVEC spheroids-laden hydrogels provided a highly suitable 3D microenvironment for bone tissue formation, which can be utilized in various applications, such as but not limited to tissue engineering, disease modeling and drug screening. STATEMENT OF SIGNIFICANCE: Stem cell encapsulation in hydrogels has been widely used in various areas such as tissue engineering, regenerative medicine, organ-on-a-chip devices and gene delivery; however, fabrication of native-like bone tissue using such an approach has been a challenge, particularly in vitro, due to the limited cell loading densities resulting in weaker cell-cell interactions and lesser extra-cellular matrix deposition. Here in this work, we have encapsulated spheroids of human mesenchymal stems cells (MSCs) in collagen/fibrin hydrogel and evaluated their viability, proliferation, osteogenic differentiation, and bone formation potential in vitro with respect to cell suspension-laden hydrogel samples. We have further incorporated human umbilical vein endothelial cells (HUVECs) into MSC spheroids and demonstrated that the presence of HUVECs in 3D spheroid culture in collagen/fibrin gel induced the formation of pre-vascular network, improved cell viability and proliferation, enhanced the osteogenic differentiation of spheroids, and increased their bone mineral deposition. In sum, MSC/HUVEC spheroids laden hydrogels provided a highly suitable 3D microenvironment for bone tissue formation, which can be utilized in various applications, such as but not limited to tissue engineering and regenerative medicine, disease modeling and drug screening.


Assuntos
Osso e Ossos/fisiologia , Colágeno/farmacologia , Fibrina/farmacologia , Células Endoteliais da Veia Umbilical Humana/citologia , Hidrogéis/farmacologia , Células-Tronco Mesenquimais/citologia , Esferoides Celulares/citologia , Engenharia Tecidual/métodos , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ratos , Esferoides Celulares/efeitos dos fármacos
18.
J Tissue Eng Regen Med ; 13(4): 664-673, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30793853

RESUMO

Blood loss remains a major concern during surgery and can increase the morbidity of the intervention. The use of topical haemostatic agents to overcome this issue therefore becomes necessary. Fibrin sealants are promising haemostatic agents due to their capacity to promote coagulation, but their effectiveness and applicability need to be improved. We have compared the haemostatic efficacy of a novel nanostructured fibrin-agarose hydrogel patch, with (c-NFAH) or without cells (a-NFAH), against two commercially available haemostatic agents in a rat model of hepatic resection. Hepatic resections were performed by making short or long incisions (mild or severe model, respectively), and haemostatic agents were applied to evaluate time to haemostasis, presence of haematoma, post-operative adhesions to adjacent tissues, and inflammation factors. We found a significantly higher haemostatic success rate (time to haemostasis) with a-NFAH than with other commercial haemostatic agents. Furthermore, other relevant outcomes investigated were also improved in the a-NFAH group, including no presence of haematoma, lower adhesions, and lower grades of haemorrhage, inflammation, and necrosis in histological analysis. Overall, these findings identify a-NFAH as a promising haemostatic agent in liver resection and likely in a range of surgical procedures.


Assuntos
Fibrina/farmacologia , Hemostáticos/farmacologia , Hidrogéis/farmacologia , Nanoestruturas/química , Sefarose/farmacologia , Animais , Hemorragia/patologia , Inflamação/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Necrose , Ratos Wistar
19.
Cell Death Dis ; 10(3): 151, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30770783

RESUMO

Colon cancer stem cells (CSCs) have been shown to be responsible for the recurrence and metastasis of colorectal cancer (CRC). As a crucial microenvironmental factor, extracellular matrix (ECM) stiffness is known to affect the stemness of CSCs. Recently, fibrin deposition in the stroma of CRC was demonstrated to be responsible for tumor development. In this study, we used salmon fibrin gel to provide a 3D ECM for colon cancer cells and investigated its effects on cell growth as well as the underlying mechanisms. Compared with stiff 420 Pascal (Pa) and 1 050 Pa gels, 90 Pa soft fibrin gel was most efficient at isolating and enriching tumor colonies. The size and number of colony formation negatively correlated with gel stiffness. Specifically, these tumor colonies exhibited efficient tumorigenicity, upregulated stem cell markers, and had anti-chemotherapeutic properties and were thus named tumor-repopulating cells (TRCs). More importantly, the self-renewal molecule Nanog was sharply induced in 3D-cultured colon TRCs; further, Nanog siRNA significantly inhibited colony formation, suggesting the indispensable role of Nanog in TRC growth. A subsequent mechanistic study illustrated that Nanog expression could be modulated through fibrin gel stiffness-induced DAB2IP/PI3K/FOXA1 signaling in colon TRCs.


Assuntos
Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Fibrina/farmacologia , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias do Colo/patologia , Fibrina/metabolismo , Géis/metabolismo , Géis/farmacologia , Células HCT116 , Células HT29 , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína Homeobox Nanog/genética , Fosfatidilinositol 3-Quinases/metabolismo , Salmão , Transfecção , Proteínas Ativadoras de ras GTPase/genética
20.
Stem Cells ; 37(5): 663-676, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30779865

RESUMO

Poor cell homing limits the efficacy of cardiac cellular therapy. The homing peptide, cysteine-arginine-glutamic acid-lysine-alanine (CREKA), targets fibrin effectively which is involved in the repair process of tissue injury. Here, we assessed if CREKA-modified stem cells had enhanced fibrin-mediated homing ability resulting in better functional recovery and structural preservation in a rat myocardial injury model. CREKA-modified mesenchymal stem cells (CREKA-MSCs) were obtained via membrane fusion with CREKA-modified liposomes. The fibrin targeting ability of CREKA-MSCs was examined both in vitro and in vivo. Under both static and flow conditions in vitro, CREKA significantly enhanced MSCs binding ability to fibrin clots (2.6- and 2.3-fold, respectively). CREKA-MSCs showed 6.5-fold higher accumulation than unmodified MSCs in injured rat myocardium one day after administration, resulting in better structural preservation and functional recovery. Fibrin is, therefore, a novel target for enhancing homing of transplanted cells to injured myocardium, and the delivery system of fibrin-targeting is on behalf of a universalizable platform technology for regenerative medicine. Stem Cells 2019;37:663-676.


Assuntos
Sistemas de Liberação de Medicamentos , Transplante de Células-Tronco Mesenquimais , Isquemia Miocárdica/terapia , Traumatismo por Reperfusão/terapia , Animais , Modelos Animais de Doenças , Fibrina/antagonistas & inibidores , Fibrina/genética , Fibrina/farmacologia , Humanos , Células-Tronco Mesenquimais/citologia , Isquemia Miocárdica/genética , Isquemia Miocárdica/patologia , Miocárdio/patologia , Nanopartículas/química , Oligopeptídeos/farmacologia , Ratos , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia
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