RESUMO
Although fibroblast growth factor 7 (FGF7) is known to promote wound healing, its mass production poses several challenges and very few studies have assessed the feasibility of producing FGF7 in cell lines such as Chinese hamster ovary (CHO) cells. Therefore, this study sought to produce recombinant FGF7 in large quantities and evaluate its wound healing effect. To this end, the FGF7 gene was transfected into CHO cells and FGF7 production was optimized. The wound healing efficacy of N-glycosylated FGF7 was evaluated in animals on days 7 and 14 post-treatment using collagen patches (CPs), FGF7-only, and CP with FGF7 (CP+FGF7), whereas an untreated group was used as the control. Wound healing was most effective in the CP+FGF7 group. Particularly, on day 7 post-exposure, the CP+FGF7 and FGF7-only groups exhibited the highest expression of hydroxyproline, fibroblast growth factor, vascular endothelial growth factor, and transforming growth factor. Epidermalization in H&E staining showed the same order of healing as hydroxyproline content. Additionally, the CP+FGF7 and FGF7-only group exhibited more notable blood vessel formation on days 7 and 14. In conclusion, the prepared FGF7 was effective in promoting wound healing and CHO cells can be a reliable platform for the mass production of FGF7.
Assuntos
Cricetulus , Fator 7 de Crescimento de Fibroblastos , Proteínas Recombinantes , Cicatrização , Animais , Células CHO , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Cicatrização/efeitos dos fármacos , Fator 7 de Crescimento de Fibroblastos/genética , Fator 7 de Crescimento de Fibroblastos/metabolismo , Humanos , Cricetinae , Hidroxiprolina/metabolismo , Transfecção , Colágeno/metabolismoRESUMO
To enhance the production of recombinant human transforming growth factor-beta1 (rhTGF-ß1) in Chinese hamster ovary (CHO) cells, rhTGF-ß1 was first characterized for endocytosis, signaling pathway, and overall maturation process. The mature rhTGF-ß1 used for clinical application was internalized into CHO cells and inhibited the growth of CHO cells in a dose-dependent manner. However, mature rhTGF-ß1 was mostly produced in the form of latent rhTGF-ß1 in cultures of recombinant CHO (rCHO) cells producing rhTGF-ß1 (CHO-rhTGF-ß1). The concentration of active mature rhTGF-ß1 in the culture supernatant of CHO-rhTGF-ß1 cells was not high enough to compromise yield. In addition, a significant amount of unprocessed precursors was produced by CHO-rhTGF-ß1 cells. Overexpression of PACEsol, a soluble form of furin, in CHO-rhTGF-ß1 cells was effective for the proteolytic cleavage of unprocessed precursors. The highest mature rhTGF-ß1 concentration (6.4 µg mL-1 ) was obtained with the PACEsol-expressing clone, which was approximately 45% higher than that of the parental clone (P < 0.01). Thus, a comprehensive understanding of the intrinsic properties of rhTGF-ß1 with respect to the overall maturation process, signaling pathway, and endocytosis is essential for effectively enhancing the production of mature rhTGF-ß1 in CHO cells.
Assuntos
Transdução de Sinais , Fator de Crescimento Transformador beta1 , Cricetinae , Animais , Humanos , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/farmacologia , Cricetulus , Células CHO , Proteínas Recombinantes/metabolismo , EndocitoseRESUMO
Cell surface heparan sulfate proteoglycan (HSPG)-mediated endocytosis results in poor yields of recombinant human bone morphogenetic proteins (rhBMPs) from CHO cell cultures. Upon incubation of rhBMP-2 and rhBMP-7 with CHO cells at 37 °C, both rhBMP-2 and rhBMP-7 bound to the cell surface HSPGs in CHO cells, but only rhBMP-2 was actively internalized into CHO cells. Cell surface HSPGs were found to serve as the main receptor for rhBMP-2 internalization. It was also found that the cell surface HSPG-mediated endocytosis of rhBMP-2 occurred through both the clathrin- and caveolin-dependent pathways. Blockage of rhBMP-2 internalization by the addition of structural analogs of HSPGs such as dextran sulfate (DS) and heparin dramatically increased rhBMP-2 production in recombinant CHO (rCHO) cell cultures. Compared to the control cultures, addition of DS (1.0 g/L) and heparin (0.2 g/L) resulted in a 22.0- and 19.0-fold increase in the maximum rhBMP-2 concentration, respectively. In contrast, the production of rhBMP-7, which was not internalized into the rCHO cells, did not dramatically increase upon addition of DS and heparin. Taken together, rhBMPs have a different fate in terms of HSPG-mediated internalization in CHO cells. HSPG-mediated endocytosis of each rhBMP should be understood individually to increase the rhBMP yield in rCHO cell cultures.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 7/metabolismo , Endocitose , Proteoglicanas de Heparan Sulfato/metabolismo , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 7/genética , Células CHO , Cricetulus , Proteoglicanas de Heparan Sulfato/genética , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Endocytic regulation serves a critical role in modulating the extracellular level of signaling molecules, such as bone morphogenetic proteins (BMPs). Unfortunately, endocytosis may result in poor yields of recombinant human BMP-4 (rhBMP-4) from Chinese hamster ovary (CHO) cell cultures. When rhBMP-4 was incubated with CHO cells, rhBMP-4 was actively internalized into cells. Cell surface bound heparan sulfate proteoglycans (HSPGs) served as the major receptors for rhBMP-4 internalization. Removal of cell surface heparan sulfate (HS) by heparinases or reduction of HSPG synthesis by knockdown of xylosyltransferase2 (xylt2) in CHO cells decreased internalization of rhBMP-4. In addition, treatment with endocytosis inhibitors (chlorpromazine, genistein, and dynasore) identified a clathrin- and dynamin-dependent endocytic pathway as the major route for rhBMP-4 internalization. To enhance product yield by minimizing rhBMP-4 internalization in recombinant CHO (rCHO) cell cultures, we have tested various strategies to reduce HSPG synthesis (knockdown of xylt2 and sodium chlorate treatment) or inhibit the binding of rhBMP-4 to cell-surface-bound HSPGs (supplementation with heparin or dextran sulfate [DS]). Among these approaches, DS, which is a linear anionic sulfated polysaccharide with similarity to HS chains, was the most effective in enhancing rhBMP-4 production in rCHO cell cultures. Compared with the control cultures, DS addition to the culture medium (1.0 g/L) resulted in 1.4-fold and 2.3-fold increases in maximum rhBMP-4 concentration in batch and fed-batch cultures, respectively. Taken together, the addition of DS, an effective competitor of HSPGs, improved rhBMP-4 production in rCHO cell cultures through blockage of rhBMP-4 internalization.
Assuntos
Proteína Morfogenética Óssea 4/biossíntese , Endocitose/genética , Técnicas de Silenciamento de Genes , Pentosiltransferases/genética , Proteínas Recombinantes/genética , Animais , Proteína Morfogenética Óssea 4/genética , Células CHO , Cricetinae , Cricetulus , Humanos , Pentosiltransferases/metabolismo , Proteínas Recombinantes/biossíntese , UDP Xilose-Proteína XilosiltransferaseRESUMO
BACKGROUND: Anterior cruciate ligament (ACL) reconstruction is the current standard of care for ACL tears. However, the results are not consistently successful; autografts or allografts have certain disadvantages; and synthetic grafts have had poor clinical results. PURPOSE: To determine if recellularization of decellularized tendons combined with mechanical stimulation in a bioreactor could replicate the mechanical properties of the native ACL and be successfully used for ACL reconstruction in vivo. STUDY DESIGN: Controlled laboratory study. METHODS: Porcine tibialis tendons were decellularized and then recellularized with human adult bone marrow-derived stem cells. Tendons were cultured in a tissue bioreactor that provided biaxial cyclic loading for up to 7 days. To reproduce mechanical stresses similar to those experienced by the ACL within the knee joint, the tendons were subjected to simultaneous tension and torsion in the bioreactor. Expression of tendon-specific genes and newly synthesized collagen and glycosaminoglycan were used to quantify the efficacy of recellularization and dynamic bioreactor culture. The ultimate tensile load to failure and stiffness of recellularized constructs were measured after dynamic stimulation. Finally, the tissue-engineered tendons were used to reconstruct the ACL in 24 pigs, and ultimate tensile load to failure and stiffness were assessed after 3 months. RESULTS: Dynamic bioreactor culture significantly increased the expression of tendon-specific genes, the quantity of newly synthesized collagen and glycosaminoglycan, and the ultimate tensile load and stiffness of recellularized tendons. After in vivo reconstruction, the ultimate tensile load and stiffness of the tissue-engineered tendons increased significantly up to 3 months after surgery and were within 80% of the ultimate tensile load of the natural ACL. CONCLUSION: This translational study indicates that recellularization and dynamic mechanical stimuli can significantly enhance matrix synthesis and ultimate tensile load of decellularized porcine tibialis tendons. This approach to tissue engineering can be very useful for ACL reconstruction and may overcome some of the disadvantages of autografts and allografts. CLINICAL RELEVANCE: Dynamic bioreactor cultivation of tissue-engineered tendons may overcome the limitations of autografts and allografts.
Assuntos
Lesões do Ligamento Cruzado Anterior/cirurgia , Reconstrução do Ligamento Cruzado Anterior , Ligamento Cruzado Anterior/cirurgia , Tendões/transplante , Engenharia Tecidual , Animais , Fenômenos Biomecânicos , Proteínas de Caenorhabditis elegans/química , Colágeno/química , Galactosiltransferases/química , Humanos , Articulação do Joelho/cirurgia , Masculino , Células-Tronco Mesenquimais/citologia , Estresse Mecânico , Suínos , Porco Miniatura , Resistência à TraçãoRESUMO
Recently, computer-designed three-dimensional (3D) printing techniques have emerged as an active research area with almost unlimited possibilities. In this study, we used a computer-designed 3D scaffold to drive new bone formation in a bone defect. Poly-L-lactide (PLLA) and bioactive ß-tricalcium phosphate (TCP) were simply mixed to prepare ink. PLLA + TCP showed good printability from the micronozzle and solidification within few seconds, indicating that it was indeed printable ink for layer-by-layer printing. In the images, TCP on the surface of (and/or inside) PLLA in the printed PLLA + TCP scaffold looked dispersed. MG-63 cells (human osteoblastoma) adhered to and proliferated well on the printed PLLA + TCP scaffold. To assess new bone formation in vivo, the printed PLLA + TCP scaffold was implanted into a full-thickness cranial bone defect in rats. The new bone formation was monitored by microcomputed tomography and histological analysis of the in vivo PLLA + TCP scaffold with or without MG-63 cells. The bone defect was gradually spontaneously replaced with new bone tissues when we used both bioactive TCP and MG-63 cells in the PLLA scaffold. Bone formation driven by the PLLA + TCP30 scaffold with MG-63 cells was significantly greater than that in other experimental groups. Furthermore, the PLLA + TCP scaffold gradually degraded and matched well the extent of the gradual new bone formation on microcomputed tomography. In conclusion, the printed PLLA + TCP scaffold effectively supports new bone formation in a cranial bone defect.
Assuntos
Regeneração Óssea/fisiologia , Impressão Tridimensional , Crânio/patologia , Alicerces Teciduais/química , Animais , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Fluorescência , Humanos , Osteogênese , Poliésteres/química , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Engenharia Tecidual , Microtomografia por Raio-XRESUMO
AIM: To evaluate the effect of exogenous recombinant human bone morphogenic protein-7 (rhBMP-7) on transforming growth factor-ß (TGF-ß)-induced epithelial mesenchymal cell transition (EMT) and assessed its antifibrotic effect via topical application. METHODS: The cytotoxic effect of rhBMP-7 was evaluated and the EMT of human corneal epithelial cells (HECEs) was induced by TGF-ß. HECEs were then cultured in the presence of rhBMP-7 and/or hyaluronic acid (HA). EMT markers, fibronectin, E-cadherin, α-smooth muscle actin (α-SMA), and matrix metaloproteinase-9 (MMP-9), were evaluated. The level of corneal fibrosis and the reepithelization rate were evaluated using a rabbit keratectomy model. Expression of α-SMA in keratocytes were quantified following treatment with different concentrations of rhBMP-7. RESULTS: Treatment with rhBMP-7 attenuated TGF-ß-induced EMT in HECEs. It significantly attenuated fibronectin secretion (31.6%; P<0.05), the α-SMA protein level (72.2%; P<0.01), and MMP-9 expression (23.6%, P<0.05) in HECEs compared with cells grown in the presence of TGF-ß alone. E-cadherin expression was significantly enhanced (289.7%; P<0.01) in the presence of rhBMP-7. Topical application of rhBMP-7 combined with 0.1% HA significantly reduced the amount of α-SMA+ cells by 43.18% (P<0.05) at a concentration of 2.5 µg/mL and by 47.73% (P<0.05) at 25 µg/mL, compared with the control group, without disturbing corneal reepithelization. CONCLUSION: rhBMP-7 attenuates TGF-ß-induced EMT in vitro, and topical application of rhBMP-7 reduces keratocyte myodifferentiation during the early wound healing stages in vivo without hindering reepithelization. Topical rhBMP-7 application as biological eye drops seems to be feasible in diseases involving TGF-ß-related corneal fibrosis with corneal reepithelization disorders.
RESUMO
This study examines the hypothesis that injectable collagen gel can be an effective carrier for recombinant human bone morphogenetic protein-2 (rhBMP-2)'s localization to the healing tendon-bone interface. In 36 mature New Zealand White rabbits, the upper long digital extensor tendon was cut and inserted into the proximal tibial bone tunnel. Then a rhBMP-2-containing collagen gel was injected into the tendon-bone tunnel interface, using a syringe. Histological and biomechanical assessments of the tendon-bone interface were conducted at 3 and 6 weeks after implantation. In vitro testing showed that the semi-viscous collagen gel at room temperature was transformed into a firm gel state at 37°C. The rhBMP-2 release profile showed that rhBMP-2 was released from the collagen gel for more than 28 days. In vivo testing showed that fibrocartilage and new bone are formed at the interface at 6 weeks after injection of rhBMP-2. On radiography, spotty calcification appeared and enthesis-like tissue was produced successfully in the tendon at 6 weeks after injection of rhBMP-2. Use of the viscous collagen gel and rhBMP-2 mixture increased the fusion rate between the bone tunnel and tissue graft. This study demonstrates that viscous collagen gel can be an effective carrier for rhBMP-2 delivery into surgical sites, and that the injectable rhBMP-2-containing collagen gel may be applied for the enhancement of tendon-bone interface healing in the future. Copyright © 2015 John Wiley & Sons, Ltd.
Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Colágeno/farmacologia , Traumatismos dos Tendões/tratamento farmacológico , Tendões/metabolismo , Tíbia/metabolismo , Animais , Proteína Morfogenética Óssea 2/química , Colágeno/química , Implantes de Medicamento , Humanos , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Traumatismos dos Tendões/metabolismo , Traumatismos dos Tendões/patologia , Tendões/patologia , Tíbia/lesõesRESUMO
Biologic augmentation for rotator cuff repair is a challenging treatment in patients with chronic large, massive, and irreparable rotator cuff injuries. Particularly, the use of an extracellular matrix (ECM) patch such as dermal tissue offered improved biomechanical properties in previous studies. Cytokines induce cell chemotaxis, proliferation, matrix synthesis, and cell differentiation. Moreover, osteoinductive growth factors such as bone morphogenetic protein-2 (BMP-2) affect the formation of new bone and fibrocartilage in lesions. However, the effects of using a dermal patch in combination with BMP-2 have not been evaluated to date, although many researchers have recognized the importance thereof. In this study, rhBMP-2-coated dermal patch (1 cm × 2 cm) isolated from human cadaveric donor was inserted in a rabbit model of chronic rotator cuff injury for in vivo evaluation. Bone mineral density and biomechanical strength were tested and histological and histomorphometric analyses were performed. The results showed that insertion of an rhBMP-2-coated acellular dermal patch not only significantly ameliorated new bone formation, it also improved biomechanical properties such as ultimate tensile strength. Thus, the use of this combination may improve the chronic rotator cuff injury-healing rate and clinical outcomes after rotator cuff repair. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1840-1846, 2017.
Assuntos
Curativos Biológicos , Proteína Morfogenética Óssea 2 , Materiais Revestidos Biocompatíveis , Lesões do Manguito Rotador/terapia , Pele , Animais , Proteína Morfogenética Óssea 2/química , Proteína Morfogenética Óssea 2/farmacocinética , Proteína Morfogenética Óssea 2/farmacologia , Doença Crônica , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacocinética , Materiais Revestidos Biocompatíveis/farmacologia , Masculino , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacologiaRESUMO
Sequentially chemical-treated bovine bone was not only evaluated by mechanical and chemical analyses but also implanted into the gluteal muscles of rats for 12 weeks to investigate potential local pathological effects and systemic toxicities. The test (chemical treated bone) and control (heat treated bone) materials were compared using scanning electron microscope (SEM), x-ray diffraction pattern, inductively coupled plasma analysis, and bending strength test. In the SEM images, the micro-porous structure of heat-treated bone was changed to sintered ceramic-like structure. The structure of bone mineral from test and control materials was analyzed as100% hydroxyapatite. The ratio of calcium (Ca) to potassium (P), the main inorganic elements, was same even though the Ca and P percentages of the control material was relatively higher than the test material. No death or critical symptoms arose from implantation of the test (chemical treated bone) and control (physiological saline) materials during 12 weeks. The implanted sites were macroscopically examined, with all the groups showing non-irritant results. Our results indicate that chemical processed bovine bone has a better mechanical property than the heat treated bone and the implantation of this material does not produce systemic or pathological toxicity.
Assuntos
Transplante Ósseo/métodos , Osso e Ossos/efeitos dos fármacos , Músculo Esquelético/cirurgia , Animais , Fenômenos Biomecânicos , Transplante Ósseo/efeitos adversos , Osso e Ossos/química , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/ultraestrutura , Nádegas , Cálcio/análise , Bovinos , Durapatita/análise , Feminino , Xenoenxertos , Temperatura Alta , Masculino , Microscopia Eletrônica de Varredura , Porosidade , Potássio/análise , Radiografia , Ratos , Ratos Sprague-Dawley , Medição de Risco , Espectrofotometria Atômica , Estresse Mecânico , Fatores de Tempo , Testes de Toxicidade Subcrônica , Transplante Heterólogo , Difração de Raios XRESUMO
BACKGROUND: Hydrolyzed polyacrylonitrile (HPAN) has attracted much attention as a hydrogel for a broad range of biomedical applications. Therefore, in this study, we prepared HPAN derivatives with controllable compositions by the radical polymerization of acrylonitrile (AN), methacrylic acid (MAA) and N-isopropylacrylamide (NIPAM) monomers. RESULTS: The prepared poly(AN-co-MAA-co-NIPAM) copolymers had different ratios of AN, MAA, and NIPAM and molecular weights ranging from 2000 to 50,000. The copolymers were prepared as films to examine their properties. The prepared copolymer films showed different solubilities, contact angles, and swelling ratios. The properties of the copolymer films were affected by the hydrophobic PAN segments and the hydrophilic PMAA or PNIPAM segments. CONCLUSION: Thus, we conclude that introducing PMAA and PNIPAM segments with different ratios and lengths into PAN segments could represent a method of controlling the hydrogel properties of copolymers.
RESUMO
To develop an appropriate drug carrier for drug delivery systems, we prepared random poly(lactide-co-glycolide-co-ε-caprolactone) (PLGC) copolymers in comparison to commercial poly(lactic acid-co-glycolic acid) (PLGA) grades. The molecular weights of PLGC copolymers varied from 20k to 90k g mol-1 in the total polyester segments, when poly-l-lactic acid (PLLA), polyglycolic acid (PGA), and polycaprolactone (PCL) compositions were kept constant. The lengths of PLGC copolymers varied from 10 : 10 : 80 to 40 : 40 : 20 in the PLLA : PGA : PCL segments, when the molecular weights of the total polyester segments were kept constant. The crystalline properties of the PLGA copolymers can be changed to amorphous by the incorporation of PCL segments. In vitro and in vivo degradation behavior can be easily tuned from a few days to a few weeks by changing the chemical composition of the PLGC copolymers. The in vivo inflammation associated with the PLGC implants was less pronounced than that associated with PLGA. In this study, as drug delivery carriers for locally implantable paclitaxel (Ptx) dosages, Ptx-loaded PLGC and PLGA films showed in vitro and in vivo Ptx release for 35 days. The orders of Ptx release showed profiles similar to those of in vitro and in vivo degradation of PLGC. Using near-infrared (NIR) fluorescence imaging, we confirmed the sustained release of NIR over an extended period from IR-780-loaded PLGC and PLGA implanted in live animals. In conclusion, we confirmed that compared to PLGA, PLGC effectively acts as a drug carrier for drug delivery systems.
RESUMO
Surface-modified titanium (Ti) samples with hydroxyapatite (HAp) and heparin (Hep)-bone morphogenetic protein-2 (BMP-2) complex (Ti/HAp/Hep/BMP-2) were prepared, and their efficacies on the enhancements of bone formation and osseointegration in vitro and in vivo were examined as compared to Ti/HAp and Ti/Hep/BMP-2. The modified surfaces were characterized by X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and contact angle goniometry. In vitro studies revealed that MG-63 human osteosarcoma cell lines grown on Ti/HAp/Hep/BMP-2 increased the amounts of alkaline phosphatase (ALP) activity, calcium deposition and the levels of OCN mRNA gene expression as compared to those grown on Ti/HAp, Ti/Hep/BMP-2 or pristine Ti. Moreover, Ti/HAp/Hep/BMP-2 exhibited higher bone volume (BV), bone volume/tissue volume (BV/TV), removal torque value and bone-implant contact (BIC) than Ti/HAp, Ti/Hep/BMP-2 or pristine Ti in vivo. Histological evaluations showed that many desirable features of bone remodelling existed at the interface between Ti/HAp/Hep/BMP-2 and the host bone. Consequently, Ti/HAp/Hep/BMP-2 may have potential for clinical use as dental or orthopaedic implants.
Assuntos
Desenvolvimento Ósseo , Proteína Morfogenética Óssea 2/química , Durapatita/química , Heparina/química , Osseointegração , Titânio/química , Fosfatase Alcalina/metabolismo , Animais , Proteína Morfogenética Óssea 2/metabolismo , Linhagem Celular Tumoral , Humanos , Técnicas In Vitro , Microscopia Eletrônica de Varredura , Osteocalcina/genética , Espectroscopia Fotoeletrônica , RNA Mensageiro/genética , Coelhos , Propriedades de SuperfícieRESUMO
Bone morphogenetic protein-7 (BMP-7) is synthesized as a precursor that requires proteolytic cleavage of the propeptide by proprotein convertases (PCs) for its functional activity. A high-level expression of BMP-7 in CHO cells (CHO-BMP-7) resulted in secretion of a mixture of inactive precursor and active BMP-7. In an effort to achieve efficient processing of BMP-7 in CHO cells, PCs responsible for cleavage of the precursors in CHO cells were characterized. Analysis of the mRNA expression levels of four PCs (furin, PACE4, PC5/6, and PC7) revealed that only furin and PC7 genes are expressed in CHO-BMP-7 cells. Specific inhibition of the PCs by hexa-D-arginine (D6R) or decanoyl-RVKR-chloromethyl ketone (RVKR-CMK) further revealed that furin is mainly responsible for the proteolytic processing of BMP-7. To identify a more efficient PC for BMP-7 processing, the four PC genes were transiently expressed in CHO-BMP-7 cells, respectively. Among these, PC5/6 was found to be the most efficient in BMP-7 processing. Stable overexpression of PC5/6ΔC, a secreted form of PC5/6, significantly improved mature BMP-7 production in CHO-BMP-7 cells. When the maximum BMP-7 concentration was obtained in the culture of CHO-BMP-7 cells, approximately 88% of BMP-7 was unprocessed. In contrast, no precursor was found in the culture of PC5/6ΔC-overexpressing cells (clone #97). Furthermore, the in vitro biological activity of the mature BMP-7 from PC5/6ΔC-overexpressing cells was comparable to that from CHO-BMP-7 cells. Taken together, the present results indicate that overexpression of PC5/6ΔC in CHO-BMP-7 cells is an efficient means of increasing the yield of BMP-7.
Assuntos
Proteína Morfogenética Óssea 7/metabolismo , Pró-Proteína Convertases/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Proteína Morfogenética Óssea 7/química , Proteína Morfogenética Óssea 7/genética , Células CHO , Cricetinae , Cricetulus , Humanos , Pró-Proteína Convertases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Sorghum is a principal cereal food in a number of parts of the world and is critical in folk medicine in Asia and Africa. However, its effects on bone are unknown. Growth hormone (GH) is a regulator of bone growth and bone metabolism. GH activates several signaling pathways, including the Janus kinase (Jak)/signal transducer and activator of transcription (STAT) pathways, thereby regulating expression of genes, including insulinlike growth factor (IGF)1. Bone morphogenetic proteins (BMPs) induce the differentiation of cells of the osteoblastic lineage, increasing the pool of IGF1 target cells, the mature osteoblasts. In the present study, the effects of Hwanggeumchal sorghum extracts (HSE) on GH signaling via the Jak/STAT pathway in osteoblasts were investigated. HSE was not observed to be toxic to osteoblastic cells and increased the expression of BMP7 and GHrelated proteins, including STAT5B, pSTAT5B, IGF1 receptor (IGF-1R), growth receptor hormone (GHR) and Jak2 in MC3T3E1 cells. In addition, HSE increased BMP7 and GHR mRNA expression in MC3T3E1 cells. The expression of HSEinduced BMP7 and GHR was inhibited by AG490, a Jak2 kinase inhibitor. The observations indicate that HSEinduced signaling is similar to GH signaling via the GHRJak2 signaling axis. Using small interference RNA (siRNA) analysis, STAT5B was found to play an essential role in HSEinduced BMP7 and GH signaling in MC3T3E1 cells. Results of the current study indicate that HSE promotes bone growth through activation of STAT5B.
Assuntos
Proteína Morfogenética Óssea 7/metabolismo , Hormônio do Crescimento/metabolismo , Janus Quinase 2/metabolismo , Extratos Vegetais/toxicidade , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sorghum/metabolismo , Animais , Proteína Morfogenética Óssea 7/genética , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Expressão Gênica/efeitos dos fármacos , Janus Quinase 2/antagonistas & inibidores , Camundongos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Extratos Vegetais/química , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptores da Somatotropina/genética , Receptores da Somatotropina/metabolismo , Fator de Transcrição STAT5/antagonistas & inibidores , Fator de Transcrição STAT5/genética , Sorghum/química , Tirfostinas/farmacologiaRESUMO
Decellularized tissues have been successfully used in tissue engineering and regenerative medicine for the purpose of removing antigens present in the cellular components. However, this decellularization technique uses ionic solutions or chemical treatments such as enzyme treatments that might damage the biophysical properties or reduce the physical strength of tissue. This study aimed to improve the strength of decellularized tissues. We designed a tissue bioreactor that can repeatedly deliver physical stimulation, such as tensile and torsional deformation, to the upper and lower parts of a tissue. To decellularized porcine Tibialis tendons, we used an enzymatic solution to remove the primary cells, and then applied ultrasonic cleansing using a combination of ionic solution and distilled water to destroy residual cells by differing from the osmotic pressure between the inside and outside of the cell membrane. The total DNA content of decellularized tissue was decreased by 77% compared with that of the original tissue and the ultimate tensile strength of the decellularized tissue was 20% lower than that of the normal tissue. Decellularized tissues were then cultivated in the tissue bioreactor with repeated physical stimulation of 110% tension, 90° torsion, and frequency of once per a second, and the ultimate tensile strength was found to be greater than that of the normal ligament at 7 day culture. This study showed that decellularization using enzyme and mechanical treatment is safe and use of a tissue bioreactor can increase the physical strength of tendons, making this a potential mechanism to reconstruct human ligaments.
Assuntos
Reatores Biológicos , Tendões/citologia , Tendões/fisiologia , Técnicas de Cultura de Tecidos/instrumentação , Animais , Fenômenos Biomecânicos , Eletroforese em Gel de Poliacrilamida , Humanos , Coloração e Rotulagem , Sus scrofa , Tendões/ultraestrutura , Resistência à Tração/fisiologiaRESUMO
Articular cartilage, when damaged by degenerative disease or trauma, has limited ability for self-repair. Recently, many trials have demonstrated that gene therapy combined with tissue engineering techniques would be a promising approach for cartilage regeneration. Bone morphogenetic protein 2 (BMP-2) is an important signal for upregulation of osteogenesis and chondrogenesis of stem cells. Sex-determining region Y box gene 9 (SOX-9) has also been reported as one of the key transcription factors for chondrogenesis. We hypothesized that codelivery of BMP-2 and SOX-9 genes would result in improved efficiency of recovery of normal chondrogenic properties in dedifferentiated chondrocytes. To this aim, we constructed a bicistronic vector encoding the BMP-2 and SOX-9 genes linked to the "self-cleaving" 2A peptide sequence. After gene delivery to dedifferentiated chondrocytes using a microporator transfection system, we confirmed over 65% delivery efficiency of the BMP-2 and SOX-9 genes. According to RT-PCR analysis and Alcian blue staining, simultaneous delivery of BMP-2/SOX-9 resulted in significantly increased expression of chondrogenesis-related markers (type II collagen and aggrecan) and GAG matrix formation compared with individual delivery of the BMP-2 or SOX-9 gene. Six weeks after in vivo transplantation, BMP-2/SOX-9 genes also showed a significant increase in cartilage formation compared with the BMP-2 or SOX-9 gene. These results demonstrate that codelivery of two chondrogenic lineage-determining genes can enhance normal chondrogenic properties of dedifferentiated chondrocytes followed by improved cartilage formation.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Cartilagem Articular/fisiologia , Condrócitos/fisiologia , Fatores de Transcrição SOX9/metabolismo , Adulto , Animais , Proteína Morfogenética Óssea 2/administração & dosagem , Proteína Morfogenética Óssea 2/biossíntese , Proteína Morfogenética Óssea 2/genética , Cartilagem Articular/crescimento & desenvolvimento , Cartilagem Articular/metabolismo , Desdiferenciação Celular/genética , Condrócitos/citologia , Condrócitos/metabolismo , Feminino , Técnicas de Transferência de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fatores de Transcrição SOX9/administração & dosagem , Fatores de Transcrição SOX9/biossíntese , Fatores de Transcrição SOX9/genética , TransfecçãoRESUMO
BACKGROUND: Xenografts, unlike other grafting products, cannot be commercialized unless they conform to stringent safety regulations. Particularly with bovine-derived materials, it is essential to remove viruses and inactivate infectious factors because of the possibility that raw materials are imbrued with infectious viruses. The removal of the characteristics of infectious viruses from the bovine bone grafting materials need to be proved and inactivation process should satisfy the management provision of the Food and Drug Administration (FDA). To date, while most virus inactivation studies were performed in human allograft tissues, there have been almost no studies on bovine bone. METHODS: To evaluate the efficacy of virus inactivation after treatment of bovine bone with 70% ethanol, 4% sodium hydroxide, and gamma irradiation, we selected a variety of experimental model viruses that are known to be associated with bone pathogenesis, including bovine parvovirus (BPV), bovine herpes virus (BHV), bovine viral diarrhea virus (BVDV), and bovine parainfluenza-3 virus (BPIV-3). The cumulative virus log clearance factor or cumulative virus log reduction factor for the manufacturing process was obtained by calculating the sum of the individual virus log clearance factors or log reduction factors determined for individual process steps with different physicochemical methods. RESULTS: The cumulative log clearance factors achieved by three different virus inactivation processes were as follows: BPV ≥ 17.73, BHV ≥ 20.53, BVDV ≥ 19.00, and BPIV-3 ≥ 16.27. On the other hand, the cumulative log reduction factors achieved were as follows: BPV ≥ 16.95, BHV ≥ 20.22, BVDV ≥ 19.27, and BPIV-3 ≥ 15.58. CONCLUSIONS: Treatment with 70% ethanol, 4% sodium hydroxide, or gamma irradiation was found to be very effective in virus inactivation, since all viruses were at undetectable levels during each process. We have no doubt that application of this established process to bovine bone graft manufacture will be effective and essential.
Assuntos
Transplante Ósseo , Vírus de DNA/efeitos dos fármacos , Vírus de DNA/efeitos da radiação , Raios gama , Transplantes/virologia , Inativação de Vírus/efeitos dos fármacos , Inativação de Vírus/efeitos da radiação , Animais , Bovinos , Linhagem Celular , Células Cultivadas , Vírus de DNA/metabolismo , Combinação de Medicamentos , HumanosRESUMO
Methylsulfonylmethane (MSM) is a naturally occurring sulfur compound with well-known anti-oxidant properties and anti-inflammatory activities. But, its effects on bone are unknown. Growth hormone (GH) is regulator of bone growth and bone metabolism. GH activates several signaling pathways such as the Janus kinase (Jak)/signal transducers and activators of transcription (STAT) pathway, thereby regulating expression of genes including insulin-like growth factor (IGF)-1. GH exerts effects both directly and via IGF-1, which signals by activating the IGF-1 receptor (IGF-1R). In this study, we investigated the effects of MSM on the GH signaling via the Jak/STAT pathway in osteoblasts and the differentiation of primary bone marrow mesenchymal stem cells (MSCs). MSM was not toxic to osteoblastic cells and MSCs. MSM increased the expression of GH-related proteins including IGF-1R, p-IGF-1R, STAT5b, p-STAT5b, and Jak2 in osteoblastic cells and MSCs. MSM increased IGF-1R and GHR mRNA expression in osteoblastic cells. The expression of MSM-induced IGF-1R and GHR was inhibited by AG490, a Jak2 kinase inhibitor. MSM induced binding of STAT5 to the IGF-1R and increased IGF-1 and IGF-1R promoter activities. Analysis of cell extracts by immunoprecipitation and Western blot showed that MSM enhanced GH-induced activation of Jak2/STAT5b. We found that MSM and GH, separately or in combination, activated GH signaling via the Jak2/STAT5b pathway in UMR-106 cells. Using siRNA analysis, we found that STAT5b plays an essential role in GH signaling activation in C3H10T1/2 cells. Osteogenic marker genes (ALP, ON, OCN, BSP, OSX, and Runx2) were activated by MSM, and siRNA-mediated STAT5b knockdown inhibited MSM-induced expression of osteogenic markers. Furthermore, MSM increased ALP activity and the mineralization of MSCs. Taken together, these results indicated that MSM can promote osteogenic differentiation of MSCs through activation of STAT5b.
Assuntos
Dimetil Sulfóxido/farmacologia , Hormônio do Crescimento/metabolismo , Janus Quinase 2/metabolismo , Osteogênese/efeitos dos fármacos , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sulfonas/farmacologia , Análise de Variância , Animais , Western Blotting , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Primers do DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Imunoprecipitação , Luciferases , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Osteoblastos/fisiologia , RNA Interferente Pequeno/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sais de Tetrazólio , TiazóisRESUMO
OBJECTIVE: The aim of the current study was to determine whether a hydroxyapatite (HA)/beta-tricalcium phosphate (ß-TCP) ratio of 20/80 impregnated with recombinant human bone morphogenetic protein (rhBMP-2) enhances new bone formation and to evaluate the dose-dependent response of rhBMP-2. STUDY DESIGN: Critical-sized calvarial defects were made in rats, and biphasic calcium phosphate (BCP) with different rhBMP-2 doses was loaded into rat calvarial defects. The animals were allowed to heal for either 2 or 8 weeks. RESULTS: The percentages of new bone after 2 and 8 weeks of healing were significantly greater in the rhBMP-2-treated groups (at all doses) than in the control groups. The percentage of remaining BCP was significantly lower at 8 weeks than at 2 weeks in all groups that included BCP. CONCLUSIONS: rhBMP-2 administered using a BCP carrier significantly induces new bone formation. A dose-dependent response was not shown in the present study.