RESUMO
Background: Bone tissue engineering (BTE) is a promising alternative to autologous bone grafting for the clinical treatment of bone defects, and inorganic/organic composite hydrogels as BTE scaffolds are a hot spot in current research. The construction of nano-hydroxyapatite/gelatin methacrylate/oxidized sodium alginate (nHAP/GelMA/OSA), abbreviated as HGO, composite hydrogels loaded with bone morphogenetic protein 7 (BMP7) will provide a suitable 3D microenvironment to promote cell aggregation, proliferation, and differentiation, thus facilitating bone repair and regeneration. Methods: Dually-crosslinked hydrogels were fabricated by combining GelMA and OSA, while HGO hydrogels were formulated by incorporating varying amounts of nHAP. The hydrogels were physically and chemically characterized followed by the assessment of their biocompatibility. BMP7-HGO (BHGO) hydrogels were fabricated by incorporating suitable concentrations of BMP7 into HGO hydrogels. The osteogenic potential of BHGO hydrogels was then validated through in vitro experiments and using rat femoral defect models. Results: The addition of nHAP significantly improved the physical properties of the hydrogel, and the composite hydrogel with 10% nHAP demonstrated the best overall performance among all groups. The selected concentration of HGO hydrogel served as a carrier for BMP7 loading and was evaluated for its osteogenic potential both in vivo and in vitro. The BHGO hydrogel demonstrated superior in vitro osteogenic induction and in vivo potential for repairing bone tissue compared to the outcomes observed in the blank control, BMP7, and HGO groups. Conclusion: Using hydrogel containing 10% HGO appears promising for bone tissue engineering scaffolds, especially when loaded with BMP7 to boost its osteogenic potential. However, further investigation is needed to optimize the GelMA, OSA, and nHAP ratios, along with the BMP7 concentration, to maximize the osteogenic potential.
Assuntos
Alginatos , Proteína Morfogenética Óssea 7 , Regeneração Óssea , Durapatita , Gelatina , Hidrogéis , Osteogênese , Engenharia Tecidual , Alicerces Teciduais , Alginatos/química , Alginatos/farmacologia , Animais , Proteína Morfogenética Óssea 7/química , Proteína Morfogenética Óssea 7/farmacologia , Gelatina/química , Engenharia Tecidual/métodos , Hidrogéis/química , Hidrogéis/farmacologia , Durapatita/química , Durapatita/farmacologia , Osteogênese/efeitos dos fármacos , Ratos , Regeneração Óssea/efeitos dos fármacos , Alicerces Teciduais/química , Ratos Sprague-Dawley , Metacrilatos/química , Masculino , Humanos , Osso e Ossos/efeitos dos fármacosRESUMO
Bone morphogenetic proteins (BMPs) are an important family of growth factors playing a role in a large number of physiological and pathological processes, including bone homeostasis, tissue regeneration, and cancers. In vivo, BMPs bind successively to both BMP receptors (BMPRs) of type I and type II, and a promiscuity has been reported. In this study, we used biolayer interferometry to perform parallel real-time biosensing and to deduce the kinetic parameters (ka, kd) and the equilibrium constant (KD) for a large range of BMP/BMPR combinations in similar experimental conditions. We selected four members of the BMP family (BMP-2, 4, 7, 9) known for their physiological relevance and studied their interactions with five type-I BMP receptors (ALK1, 2, 3, 5, 6) and three type-II BMP receptors (BMPR-II, ACTR-IIA, ACTR-IIB). We reveal that BMP-2 and BMP-4 behave differently, especially regarding their kinetic interactions and affinities with the type-II BMPR. We found that BMP-7 has a higher affinity for the type-II BMPR receptor ACTR-IIA and a tenfold lower affinity with the type-I receptors. While BMP-9 has a high and similar affinity for all type-II receptors, it can interact with ALK5 and ALK2, in addition to ALK1. Interestingly, we also found that all BMPs can interact with ALK5. The interaction between BMPs and both type-I and type-II receptors in a ternary complex did not reveal further cooperativity. Our work provides a synthetic view of the interactions of these BMPs with their receptors and paves the way for future studies on their cell-type and receptor specific signaling pathways.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Técnicas Biossensoriais/métodos , Proteína Morfogenética Óssea 2/química , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/química , Proteína Morfogenética Óssea 4/metabolismo , Proteína Morfogenética Óssea 7/química , Proteína Morfogenética Óssea 7/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas/química , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/química , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Proteínas Morfogenéticas Ósseas/química , Dimerização , Fator 2 de Diferenciação de Crescimento/química , Fator 2 de Diferenciação de Crescimento/metabolismo , Humanos , Interferometria , Cinética , Ligação Proteica , Ressonância de Plasmônio de SuperfícieAssuntos
Proteína Morfogenética Óssea 2/química , Proteína Morfogenética Óssea 7/química , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/química , Multimerização Proteica , Proteínas de Peixe-Zebra/química , Peixe-Zebra/metabolismo , Animais , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 7/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Proteínas de Peixe-Zebra/metabolismoRESUMO
Since their discovery as pluripotent cytokines extractable from bone matrix, it has been speculated how bone morphogenetic proteins (BMPs) become released and activated from the extracellular matrix (ECM). In contrast to TGF-ßs, most investigated BMPs are secreted as bioactive prodomain (PD)-growth factor (GF) complexes (CPLXs). Recently, we demonstrated that PD-dependent targeting of BMP-7 CPLXs to the extracellular fibrillin microfibril (FMF) components fibrillin-1 and -2 represents a BMP sequestration mechanism by rendering the GF latent. Understanding how BMPs become activated from ECM scaffolds such as FMF is crucial to elucidate pathomechanisms characterized by aberrant BMP activation and ECM destruction. Here, we describe a new MMP-dependent BMP-7 activation mechanism from ECM-targeted pools via specific PD degradation. Using Edman sequencing and mutagenesis, we identified a new and conserved MMP-13 cleavage site within the BMP-7 PD. A degradation screen with different BMP family PDs and representative MMP family members suggested utilization of the identified site in a general MMP-driven BMP activation mechanism. Furthermore, sandwich ELISA and solid phase cleavage studies in combination with bioactivity assays, single particle TEM, and in silico molecular docking experiments provided evidence that PD cleavage by MMP-13 leads to BMP-7 CPLX disintegration and bioactive GF release.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Matriz Extracelular/metabolismo , Metaloproteinases da Matriz/fisiologia , Motivos de Aminoácidos , Animais , Proteína Morfogenética Óssea 7/química , Proteína Morfogenética Óssea 7/metabolismo , Proteínas Morfogenéticas Ósseas/química , Células HEK293 , Humanos , Metaloproteinase 13 da Matriz/fisiologia , Camundongos , Simulação de Acoplamento Molecular , Domínios ProteicosRESUMO
While the bone morphogenetic protein-7 (BMP-7) is a well-known therapeutic growth factor reverting many fibrotic diseases, including peritoneal fibrosis by peritoneal dialysis (PD), soluble growth factors are largely limited in clinical applications owing to their short half-life in clinical settings. Recently, we developed a novel drug delivery model using protein transduction domains (PTD) overcoming limitation of soluble recombinant proteins, including bone morphogenetic protein-7 (BMP-7). This study aims at evaluating the therapeutic effects of PTD-BMP-7 consisted of PTD and full-length BMP-7 on epithelial-mesenchymal transition (EMT)-related fibrosis. Human peritoneal mesothelial cells (HPMCs) were then treated with TGF-ß1 or TGF-ß1 + PTD-BMP-7. Peritoneal dialysis (PD) catheters were inserted into Sprague-Dawley rats, and these rats were infused intra-peritoneally with saline, peritoneal dialysis fluid (PDF) or PDF + PTD-BMP-7. In vitro, TGF-ß1 treatment significantly increased fibronectin, type I collagen, α-SMA and Snail expression, while reducing E-cadherin expression in HPMCs (P < .001). PTD-BMP-7 treatment ameliorated TGF-ß1-induced fibronectin, type I collagen, α-SMA and Snail expression, and restored E-cadherin expression in HPMCs (P < .001). In vivo, the expressions of EMT-related molecules and the thickness of the sub-mesothelial layer were significantly increased in the peritoneum of rats treated with PDF, and these changes were significantly abrogated by the intra-peritoneal administration of PTD-BMP-7. PTD-BMP-7 treatment significantly inhibited the progression of established PD fibrosis. These findings suggest that PTD-BMP-7, as a prodrug of BMP-7, can be an effective therapeutic agent for peritoneal fibrosis in PD patients.
Assuntos
Proteína Morfogenética Óssea 7/administração & dosagem , Sistemas de Liberação de Medicamentos , Fibrose Peritoneal/tratamento farmacológico , Animais , Biomarcadores , Proteína Morfogenética Óssea 7/química , Modelos Animais de Doenças , Desenho de Fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Humanos , Imuno-Histoquímica , Microscopia Intravital , Masculino , Camundongos , Fibrose Peritoneal/etiologia , Fibrose Peritoneal/metabolismo , Fibrose Peritoneal/patologia , Ratos , Proteínas Recombinantes , Resultado do TratamentoRESUMO
The adsorption/desorption behavior, and conformational and orientational changes of proteins on the surface of biomaterials are significant parameters for understanding how biomaterials perform their biological functions. In this study, for the first time, the interactions between BMP-7 and ß-TCP (001) surface models with different ion-rich terminations (Ca-rich and P-rich) were investigated by molecular dynamics simulation (MD) and steered molecular dynamics simulation (SMD). The results indicated that BMP-7 preferentially interacts with both Ca-rich and P-rich ß-TCP (001) surfaces at its wrist epitope residues with certain conformational changes, which led to more exposure of BMP-7 knuckle epitope residues to the environment and facilitation for binding to the type II receptor. Compared to the P-rich surface, it is speculated that the Ca-rich surface was more conducive to BMP-7 signal transduction since the upright orientation of the protein adsorption would lead to smaller hindrance for receptor binding. This study provided more atomistic and molecular information for better understanding the process of Ca-P surfaces affecting BMP-7 biological properties and further interpreted the osteoinductive mechanism from the perspective of growth factor adsorption. Moreover, the docking screening method adopted in this study is of guiding significance to the design and development of bioactive materials.
Assuntos
Proteína Morfogenética Óssea 7/metabolismo , Fosfatos de Cálcio/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteína Morfogenética Óssea 7/química , Ligação ProteicaRESUMO
In contrast to the early acting bone morphogenetic protein 2, bone morphogenetic protein 7 (BMP7) plays a decisive role mainly in the late stages of bone formation. To overcome deactivation and degradation of expensive BMP7, we designed a novel long-acting BMP7 release system based on poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB) nanoparticles to enable the induction of osteogenic differentiation in human adipose mesenchymal stem cells (ADSCs). In order to improve the encapsulation efficiency of BMP7 and avoid damage by organic solvents, BMP7 was modified and protected using the biosurfactant soybean lecithin. In an in vitro test, BMP7-soybean lecithin-P34HB nanoparticles (BMP7-SPNPs) showed a short initial burst of BMP7 release during the first 24h, followed by a steady increase to a cumulative 80% release in 20days. Compared with the rapid release of control P34HB nanoparticles without soybean phospholipids loaded with BMP7 without soybean lecithin, BMP7-SPNPs significantly reduced the initial burst of BMP7 release and stabilized the content of BMP7 to allow long-term osteogenic differentiation during the late phase of bone development. Human ADSCs treated with BMP7-SPNPs showed higher alkaline phosphatase activity and higher expression levels of genetic markers of osteogenic differentiation compared with the control group. Thus, the results indicate that BMP7-SPNPs can be used as a rapid and long-acting BMP7 delivery system for osteogenic differentiation.
Assuntos
Tecido Adiposo/metabolismo , Proteína Morfogenética Óssea 7 , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Nanopartículas , Osteogênese/efeitos dos fármacos , Proteína Morfogenética Óssea 7/química , Proteína Morfogenética Óssea 7/farmacocinética , Proteína Morfogenética Óssea 7/farmacologia , Linhagem Celular , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Preparações de Ação Retardada/farmacologia , Humanos , Nanopartículas/química , Nanopartículas/uso terapêuticoAssuntos
Proteína Morfogenética Óssea 2/química , Regeneração Óssea/efeitos dos fármacos , Substitutos Ósseos , Fêmur/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 7/química , Modelos Animais de Doenças , Cães , Sistemas de Liberação de Medicamentos , Feminino , Fatores de Crescimento Neural/química , Osteogênese/efeitos dos fármacos , Oxigênio/química , Porosidade , Alicerces Teciduais/químicaRESUMO
Brown adipocytes coordinate systemic energy metabolism associated with the pathogenesis of obesity and related metabolic diseases including type 2 diabetes. We have previously reported chemical compound-induced brown adipocytes (ciBAs) converted from human dermal fibroblasts without using transgenes. In this study, to reveal a precise molecular mechanism underlying the direct conversion and human adipocyte browning, we developed serum-free brown adipogenic medium (SFBAM) with an optimized chemical cocktail consisting of Rosiglitazone, Forskolin, and BMP7. During the direct conversion, treatment with BMP7 enhanced Ucp1 expression rather than the conversion efficiency in the absence of BMP signalling inhibitors. Moreover, treatment with a TGF-ß signalling pathway inhibitor was no longer required in the serum-free medium, likely because the TGF-ß pathway was already suppressed. SFBAM and the chemical cocktail efficiently converted human dermal fibroblasts into ciBAs within four weeks. The ciBAs exhibited increased mitochondrial levels, elevated oxygen consumption rate, and a response to ß-adrenergic receptor agonists. Thus the ciBAs converted by the serum-free medium and the chemical cocktail provide a novel model of human brown (beige) adipocytes applicable for basic research, drug screening, and clinical applications.
Assuntos
Adipócitos Marrons/metabolismo , Diferenciação Celular , Derme/metabolismo , Fibroblastos/metabolismo , Transdução de Sinais , Adipócitos Marrons/citologia , Proteína Morfogenética Óssea 7/química , Proteína Morfogenética Óssea 7/farmacologia , Colforsina/química , Colforsina/farmacologia , Meios de Cultura Livres de Soro/química , Meios de Cultura Livres de Soro/farmacologia , Derme/citologia , Fibroblastos/citologia , Humanos , Rosiglitazona/química , Rosiglitazona/farmacologiaRESUMO
Numerous bioactive molecules produced in cells are involved in the process of bone formation. We consider that appropriate, simultaneous application of two types of bioactive molecules would accelerate the regeneration of tissues and organs. Therefore, we combined aspirin-loaded liposomes (Asp@Lipo) and bone forming peptide-1 (BFP-1) on three dimensional-printed polycaprolactone (PCL) scaffold and determined whether this system improved bone regeneration outcomes. in vitro experiments indicated that Asp@Lipo/BFP-1at a 3:7 ratio was the best option for enhancing the osteogenic efficiency of human mesenchymal stem cells (hMSCs). This was confirmed in an in vivo cranial defect animal model. In addition, RNA-Seq was applied for preliminarily exploration of the mechanism of action of this composite scaffold system, and the results suggested that it mainly improved bone regeneration via the PI3K/AKT signaling pathway. This approach will have potential for application in bone tissue engineering and regenerative medicine.
Assuntos
Proteína Morfogenética Óssea 7/química , Osso e Ossos/química , Fragmentos de Peptídeos/química , Poliésteres/química , Alicerces Teciduais/química , Animais , Anti-Inflamatórios não Esteroides/química , Aspirina/química , Proteína Morfogenética Óssea 7/farmacologia , Regeneração Óssea , Proliferação de Células , Células Cultivadas , Humanos , Lipossomos/química , Células-Tronco Mesenquimais , Modelos Animais , Osteogênese , Fragmentos de Peptídeos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Impressão Tridimensional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Coelhos , Transdução de Sinais , Crânio , Engenharia Tecidual , Microtomografia por Raio-XRESUMO
Bone morphogenetic protein-7 is (BMP-7) is a potent anti-inflammatory growth factor belonging to the Transforming Growth Factor Beta (TGF-ß) superfamily. It plays an important role in various biological processes, including embryogenesis, hematopoiesis, neurogenesis and skeletal morphogenesis. BMP-7 stimulates the target cells by binding to specific membrane-bound receptor BMPR 2 and transduces signals through mothers against decapentaplegic (Smads) and mitogen activated protein kinase (MAPK) pathways. To date, rhBMP-7 has been used clinically to induce the differentiation of mesenchymal stem cells bordering the bone fracture site into chondrocytes, osteoclasts, the formation of new bone via calcium deposition and to stimulate the repair of bone fracture. However, its use in cardiovascular diseases, such as atherosclerosis, myocardial infarction, and diabetic cardiomyopathy is currently being explored. More importantly, these cardiovascular diseases are associated with inflammation and infiltrated monocytes where BMP-7 has been demonstrated to be a key player in the differentiation of pro-inflammatory monocytes, or M1 macrophages, into anti-inflammatory M2 macrophages, which reduces developed cardiac dysfunction. Therefore, this review focuses on the molecular mechanisms of BMP-7 treatment in cardiovascular disease and its role as an anti-fibrotic, anti-apoptotic and anti-inflammatory growth factor, which emphasizes its potential therapeutic significance in heart diseases.
Assuntos
Proteína Morfogenética Óssea 7/metabolismo , Cardiopatias/metabolismo , Inflamação/metabolismo , Sequência de Aminoácidos , Animais , Proteína Morfogenética Óssea 7/química , Cardiopatias/patologia , Humanos , Inflamação/patologia , Modelos Biológicos , Transdução de SinaisRESUMO
This study analyzed the influence of titanium (TiO2 ) surface modifications with two osteogenic proteins (BMP-2, BMP-7) and an anti-osteoclastic drug (alendronic acid [AA]) on sandblasted/acid-etched (SLA) and plain TiO2 (PT) on cell adhesion, proliferation and differentiation (alkaline phosphatase [AP] and osteocalcin [OC]) of bone-marrow derived stem cells (BMSCs) after 1, 3 and 7 days in-vitro. Initially, AA surfaces showed the highest cell number and surface coverage. At day 3 and 7, BMP and AA-modified surfaces exhibited a significantly enhanced cell growth. For proliferation, at days 3 and 7, an enhancement on BMP-2, BMP-7 and AA-surfaces was seen. At day 7, SLA also showed a higher proliferation when compared to PT. Initially, AP expression was elevated on SLA and AA surfaces. At days 3 and 7, a significant increased AP expression was seen for SLA, BMP-2, BMP-7 and AA discs. For OC, SLA and AA surfaces had the highest expression after 1 day whereas after 3 and 7 days a significant difference was recorded for SLA, BMP-2, BMP-7 and AA. In conclusion, a beneficial biological effect of a chemical immobilization method of BMP-2, BMP-7 and alendronate onto titanium surfaces on BMSCs was proven.
Assuntos
Alendronato/farmacologia , Materiais Biocompatíveis/farmacologia , Proteína Morfogenética Óssea 2/farmacologia , Proteína Morfogenética Óssea 7/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Alendronato/química , Materiais Biocompatíveis/química , Conservadores da Densidade Óssea/química , Conservadores da Densidade Óssea/farmacologia , Células da Medula Óssea/citologia , Proteína Morfogenética Óssea 2/química , Proteína Morfogenética Óssea 7/química , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/farmacologia , Osteogênese/efeitos dos fármacos , Células-Tronco/citologia , Propriedades de Superfície , Titânio/química , Titânio/farmacologiaRESUMO
In this study, porous chitosan/collagen scaffolds were prepared through a freeze-drying process, and loaded with the plasmid vector encoding human bone morphogenetic protein-7 (BMP-7) gene. To investigate the feasibility and efficacy of this gene-activated scaffold on dental tissue engineering, human dental pulp stem cells (DPSCs) were seeded in this scaffold for in vitro and in vivo study. In vitro results indicated that cells can be transfected successfully by loaded plasmid and secrete BMP-7 until day 24. Evaluation of DNA content, ALP activity, calcium content, SEM, and real-time PCR revealed that cells on gene-activated scaffold showed better proliferation properties and odontoblastic differentiation behaviors than cells on pure scaffolds. Then, these cell-scaffold complexes were implanted subcutaneously and retrieved after 4 weeks for histology evaluation. In vivo results that gene-activated scaffold group could still trace the existence of tranfected cells at week 4 and showed the upregulated expression of DSPP compared to pure scaffold groups. On the basis of our results, chitosan/collagen-loaded BMP-7 DNA appears to be an effective substrate candidate for gene delivery and indeed enhanced DPSCs differentiation toward an odontoblast-like phenotype in vitro and in vivo. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.
Assuntos
Proteína Morfogenética Óssea 7 , Diferenciação Celular/efeitos dos fármacos , Colágeno/química , DNA , Polpa Dentária/metabolismo , Odontoblastos/metabolismo , Células-Tronco/metabolismo , Adolescente , Adulto , Animais , Proteína Morfogenética Óssea 7/química , Proteína Morfogenética Óssea 7/farmacologia , DNA/química , DNA/farmacologia , Feminino , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Células-TroncoRESUMO
The surface modification of polymeric materials has become critical for improving the bone repair capability of materials. In this study, we used a poly-L-lysine (PLL) coating method to prepare functional poly (lactic acid-glycolic acid) (PLGA) cell microcarriers, and bone morphogenetic protein 7 (BMP-7) and ponericin G1 were immobilized on the surface of microcarriers. The scanning electron microscopy (SEM), water contact angle measurement, and energy-dispersive X-ray spectroscopy (EDX) was used to analyse the surface morphology of PLL-modified PLGA microcarriers (PLL@PLGA) and their ability to promote mineralization. At the same time, the growth factor binding efficiency and antimicrobial activity of the microcarriers were studied. The effects of microcarriers on cell behaviors were evaluated by cultivating MC3T3-E1 cells on different microcarriers. The results showed that the hydrophilicity, protein adsorption, and mineralization induction capability of the microcarriers were significantly improved by PLL surface modification. The biological experiments revealed that BMP-7 and ponericin G1 immobilized-PLL modified microcarriers can effectively inhibit the proliferation of pathogenic microorganisms while enhancing the ability of the microcarriers to promote cell proliferation and osteogenesis differentiation. Therefore, we believe that PLL-modified PLGA cell microcarriers loaded with BMP-7 and ponericin G1 (PLL@PLGA/BMP-7/ponericin G1) have great potential in the field of bone repair.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Osteogênese/efeitos dos fármacos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacologia , Polilisina/química , Células 3T3 , Adsorção , Animais , Proteína Morfogenética Óssea 7/química , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Camundongos , Alicerces Teciduais/químicaRESUMO
Delivery of synovium-resident mesenchymal stem cells (synMSCs) to cartilage defect site might provide a novel therapeutic modality for treatment of articular cartilage diseases. However, low isolation efficiency of synMSCs limits their therapeutic application. Niche-preserving non-enzymatic isolation of synMSCs was firstly attempted by employing micro-organ culture system based on recapitulating tissue-specific homeostasis ex vivo. The isolated synMSCs retained superior long-term growth competency, proliferation and chondrogenic potential to bone marrow-derived MSCs (BMSCs). It was noted that synMSCs demonstrated 9-fold increase in cartilaginous micro-tissue formation and 13-fold increase in sulfated proteoglycans deposition compared to BMSCs. For delivery of synMSCs, fibrous PLGA scaffolds were specifically designed for full-thickness osteochondral defects in rabbits. The scaffolds provided effective micro-environment for growth and host-integration of synMSCs. Combined delivery of synMSCs with bone morphogenetic proteins-7 (BMP-7) was designed to achieve synergistic therapeutic efficacy. BMP-7-loaded PLGA nanoparticles electrosprayed onto the scaffolds released BMP-7 over 2â¯weeks to conform with its aimed role in stimulating early stage endochondral ossification. Scaffold-supported combined administration of synMSCs with BMP-7 resulted in high proteoglycan and collagen type II induction and thick hyaline cartilage formation. Intra-articular co-delivery of synMSCs with BMP-7 via fibrous PLGA scaffolds may be a promising therapeutic modality for articular cartilage repair.
Assuntos
Proteína Morfogenética Óssea 7/química , Cartilagem Articular/efeitos dos fármacos , Portadores de Fármacos/química , Células-Tronco Mesenquimais/metabolismo , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Membrana Sinovial/química , Animais , Medula Óssea/metabolismo , Proteína Morfogenética Óssea 7/farmacocinética , Regeneração Óssea/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Colágeno Tipo II/metabolismo , Liberação Controlada de Fármacos , Fibrina/química , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Injeções Intra-Articulares , Masculino , Transplante de Células-Tronco Mesenquimais , Osteogênese/efeitos dos fármacos , Proteoglicanas/metabolismo , Coelhos , Engenharia Tecidual , Alicerces Teciduais/químicaRESUMO
BACKGROUND: Pleural fibrosis is defined as excessive depositions of matrix components that result in pleural tissue architecture destruction and dysfunction. In severe cases, the progression of pleural fibrosis leads to lung entrapment, resulting in dyspnea and respiratory failure. However, the mechanism of pleural fibrosis is poorly understood. METHODS: miR-4739 levels were detected by miRNA array and real-time PCR. Real-time PCR, western blotting and immunofluorescence were used to identify the expression profile of indicators related to fibrosis. Target gene of miR-4739 and promoter activity assay was measured by using dual-luciferase reporter assay system. In vivo, pleural fibrosis was evaluated by Masson staining and miR-4739 level was detected by In situ hybridization histochemistry. FINDINGS: We found that bleomycin induced up-regulation of miR-4739 in pleural mesothelial cells (PMCs). Over-regulated miR-4739 mediated mesothelial-mesenchymal transition and increased collagen-I synthesis in PMCs. Investigation on the clinical specimens revealed that high levels of miR-4739 and low levels of bone morphogenetic protein 7 (BMP-7) associated with pleural fibrosis in patients. Then we next identified that miR-4739 targeted and down-regulated BMP-7 which further resulted in unbalance between Smad1/5/9 and Smad2/3 signaling. Lastly, in vivo studies revealed that miR-4739 over-expression induced pleural fibrosis, and exogenous BMP-7 prevented pleural fibrosis in mice. INTERPRETATION: Our data indicated that miR-4739 targets BMP-7 which mediates pleural fibrosis. The miR-4739/BMP-7 axis is a promising therapeutic target for the disease. FUND: The National Natural Science Foundation of China.
Assuntos
Proteína Morfogenética Óssea 7/metabolismo , MicroRNAs/metabolismo , Regiões 3' não Traduzidas , Animais , Antagomirs/metabolismo , Bleomicina/farmacologia , Proteína Morfogenética Óssea 7/química , Proteína Morfogenética Óssea 7/genética , Colágeno Tipo I/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fibrose , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Pleura/citologia , Regiões Promotoras Genéticas , Ratos , Proteína Smad1/genética , Proteína Smad1/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
The aim of this study was to prepare an injectable DNA-loaded nano-calcium phosphate paste that is suitable as bioactive bone substitution material. For this we used the well-known potential of calcium phosphate in bone contact and supplemented it with DNA for the in-situ transfection of BMP-7 and VEGF-A in a critical-size bone defect. 24 New Zealand white rabbits were randomly divided into two groups: One group with BMP-7- and VEGF-A-encoding DNA on calcium phosphate nanoparticles and a control group with calcium phosphate nanoparticles only. The bone defect was created at the proximal medial tibia and filled with the DNA-loaded calcium phosphate paste. As control, a bone defect was filled with the calcium phosphate paste without DNA. The proximal tibia was investigated 2, 4 and 12 weeks after the operation. A histomorphological analysis of the dynamic bone parameters was carried out with the Osteomeasure system. The animals treated with the DNA-loaded calcium phosphate showed a statistically significantly increased bone volume per total volume after 4 weeks in comparison to the control group. Additionally, a statistically significant increase of the trabecular number and the number of osteoblasts per tissue area were observed. These results were confirmed by radiological analysis. The DNA-loaded bone paste led to a significantly faster healing of the critical-size bone defect in the rabbit model after 4 weeks. After 12 weeks, all defects had equally healed in both groups. No difference in the quality of the new bone was found. The injectable DNA-loaded calcium phosphate paste led to a faster and more sustained bone healing and induced an accelerated bone formation after 4 weeks. The material was well integrated into the bone defect and new bone was formed on its surface. The calcium phosphate paste without DNA led to a regular healing of the critical-size bone defect, but the healing was slower than the DNA-loaded paste. Thus, the in-situ transfection with BMP-7 and VEGF-A significantly improved the potential of calcium phosphate as pasty bone substitution material.
Assuntos
Materiais Biocompatíveis/química , Proteína Morfogenética Óssea 7/química , Regeneração Óssea , Fosfatos de Cálcio/química , Nanoestruturas/química , Fator A de Crescimento do Endotélio Vascular/química , Animais , Substitutos Ósseos , DNA/química , Humanos , Osteoclastos/citologia , Coelhos , Tíbia/patologia , Alicerces Teciduais , Transfecção , CicatrizaçãoRESUMO
Defects in bone are some of the most difficult injuries to treat. Biomimetic scaffolds represent a promising approach for successful bone tissue regeneration. In this study, a three-dimensional (3D) scaffold with osteo-inductive functionality was designed and assayed both in-vitro and in-vivo. Bone formation peptide-1 (BFP1), an osteo-promoting specific peptide, was covalently bound to a 3D printed polycaprolactone (PCL) scaffold using polydopamine (DOPA). The amount of BFP1 immobilized on the surface was found to increase depending on the BFP1 concentration of the loading solution. To observe the biological effects of the 3D scaffolds, human tonsil-derived mesenchymal stem cells (hTMSCs) were isolated. The cells were cultured on the scaffolds and observed to rapidly differentiate into osteoblast-like cells with osteo-promoting capabilities. The scaffolds were implanted in a rabbit calvarial defect model for 8â¯weeks and successfully stimulated both vessel and bone regeneration. Osteo-promoting 3D scaffolds may provide a safer and more efficient approach for bone repair and remodelling in regenerative medicine.
Assuntos
Proteína Morfogenética Óssea 7/química , Proteína Morfogenética Óssea 7/farmacologia , Regeneração Óssea/efeitos dos fármacos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Impressão Tridimensional , Alicerces Teciduais/química , Animais , Proteína Morfogenética Óssea 7/síntese química , Células Cultivadas , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Osteogênese/efeitos dos fármacos , Fragmentos de Peptídeos/síntese química , CoelhosRESUMO
Renal failures treatment has been faced with several problems during the last decades. Kidney tissue engineering has been created many hopes to improve treatment procedures with scaffold fabrication that can modulate kidney cells/stem cells migration to the lesion site and increase the survival of these cells at that site with imitating the role of the kidney extracellular matrix. In this study, bone morphogenetic protein-7 (BMP7) as a vital factor for kidney development and regeneration was incorporated in the polycaprolactone (PCL) nanofibers and after morphological, mechanical, and biocompatible characterization, proliferation, and survival of the human embryonic kidney cells (HEK) were investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, and gene expression while cultured on scaffolds. Mechanical properties of the PCL nanofibers modulated after combining with BMP7 and hydration degree, protein adsorption and cell adhesion were enhanced in PCL-BMP7 compared to the pure PCL. Proliferation rate and growth increased significantly in HEK cells cultured on PCL-BMP7 when compared with that of PCL and tissue culture plate, whereas these data were also confirmed via significant decrease in apoptotic genes expression level in HEK cell cultured on PCL-BMP7. According to the results, PCL-BMP7 demonstrated positive effects on the survival and proliferation rate of the kidney cells and showed has also a great potential to use as a bioimplant for kidney tissue engineering applications.
Assuntos
Proteína Morfogenética Óssea 7 , Proliferação de Células/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Rim/metabolismo , Poliésteres/química , Alicerces Teciduais/química , Proteína Morfogenética Óssea 7/química , Proteína Morfogenética Óssea 7/farmacocinética , Proteína Morfogenética Óssea 7/farmacologia , Sobrevivência Celular , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Preparações de Ação Retardada/farmacologia , Embrião de Mamíferos/citologia , Células HEK293 , Humanos , Rim/citologiaRESUMO
BACKGROUND: The Pacific abalone, Haliotis discus hannai, is the most important cultivated abalone in China. Improving abalone muscle growth and increasing the rate of growth are important genetic improvement programs in this industry. MicroRNAs are important small noncoding RNA molecules that regulate post-transcription gene expression. However, no miRNAs have been reported to regulate muscle growth in H. discus hannai. RESULTS: we profiled six small RNA libraries for three large abalone individuals (L_HD group) and three small individuals (S_HD group) using RNA sequencing technology. A total of 205 miRNAs, including 200 novel and 5 known miRNAs, were identified. In the L_HD group, 3 miRNAs were up-regulated and 7 were down-regulated compared to the S_HD specimens. Bioinformatics analysis of miRNA target genes revealed that miRNAs participated in the regulation of cellular metabolic processes, the regulation of biological processes, the Wnt signaling pathway, ECM-receptor interaction, and the MAPK signaling pathway, which are associated with regulating growth. Bone morphogenetic protein 7 (BMP7) was verified as a target gene of hdh-miR-1984 by a luciferase reporter assay and we examined the expression pattern in different developmental stages. CONCLUSION: This is the first study to demonstrate that miRNAs are related to the muscle growth of H. discus hannai. This information could be used to study the mechanisms of abalone muscle growth. These DE-miRNAs may be useful as molecular markers for functional genomics and breeding research in abalone and closely related species.