RESUMO
Biomaterial substrates composed of semi-aligned electrospun fibers are attractive supports for the regeneration of connective tissues because the fibers are durable under cyclic tensile loads and can guide cell adhesion, orientation, and gene expression. Previous studies on supported electrospun substrates have shown that both fiber diameter and mechanical deformation can independently influence cell morphology and gene expression. However, no studies have examined the effect of mechanical deformation and fiber diameter on unsupported meshes. Semi-aligned large (1.75 µm) and small (0.60 µm) diameter fiber meshes were prepared from degradable elastomeric poly(esterurethane urea) (PEUUR) meshes and characterized by tensile testing and scanning electron microscopy (SEM). Next, unsupported meshes were aligned between custom grips (with the stretch axis oriented parallel to axis of fiber alignment), seeded with C3H10T1/2 cells, and subjected to a static load (50 mN, adjusted daily), a cyclic load (4% strain at 0.25 Hz for 30 min, followed by a static tensile loading of 50 mN, daily), or no load. After 3 days of mechanical stimulation, confocal imaging was used to characterize cell shape, while measurements of deoxyribonucleic acid (DNA) content and messenger ribonucleic acid (mRNA) expression were used to characterize cell retention on unsupported meshes and expression of the connective tissue phenotype. Mechanical testing confirmed that these materials deform elastically to at least 10%. Cells adhered to unsupported meshes under all conditions and aligned with the direction of fiber orientation. Application of static and cyclic loads increased cell alignment. Cell density and mRNA expression of connective tissue proteins were not statistically different between experimental groups. However, on large diameter fiber meshes, static loading slightly elevated tenomodulin expression relative to the no load group, and tenascin-C and tenomodulin expression relative to the cyclic load group. These results demonstrate the feasibility of maintaining cell adhesion and alignment on semi-aligned fibrous elastomeric substrates under different mechanical conditions. The study confirms that cell morphology is sensitive to the mechanical environment and suggests that expression of select connective tissue genes may be enhanced on large diameter fiber meshes under static tensile loads.
Assuntos
Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Elasticidade , Células-Tronco Mesenquimais/efeitos dos fármacos , Poliuretanos/química , Poliuretanos/farmacologia , Animais , Contagem de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Teste de Materiais , Proteínas de Membrana/genética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Estresse Mecânico , Propriedades de Superfície , Tenascina/genética , Resistência à Tração , Suporte de CargaRESUMO
Although bone-patellar tendon-bone (B-PT-B) autografts are the gold standard for repair of anterior cruciate ligament ruptures, they suffer from drawbacks such as donor site morbidity and limited supply. Engineered tissues modeled after B-PT-B autografts are promising alternatives because they have the potential to regenerate connective tissue and facilitate osseointegration. Towards the long-term goal of regenerating ligaments and their bony insertions, the objective of this study was to construct 2D meshes and 3D cylindrical composite scaffolds - possessing simultaneous region-wise differences in fiber orientation, diameter, chemistry and mechanical properties - by electrospinning two different polymers from off-set spinnerets. Using a dual drum collector, 2D meshes consisting of an aligned polycaprolactone (PCL) fiber region, randomly oriented poly(lactide-co-glycolide) (PLGA) fiber region and a transition region (comprised of both PCL and PLGA fibers) were prepared, and region-wise differences were confirmed by microscopy and tensile testing. Bone marrow stromal cells (BMSCs) cultured on these meshes exhibited random orientations and low aspect ratios on the random PLGA regions, and high aspect ratios and alignment on the aligned PCL regions. Next, meshes containing an aligned PCL region flanked by two transition regions and two randomly oriented PLGA regions were prepared and processed into 3D cylindrical composite scaffolds using an interpenetrating photo-crosslinkable polyethylene glycol diacrylate hydrogel to recapitulate the shape of B-PT-B autografts. Tensile testing indicated that cylindrical composites were mechanically robust, and eventually failed due to stress concentration in the aligned PCL region. In summary, this study demonstrates a process to fabricate electrospun meshes possessing region-wise differences in properties that can elicit region-dependent cell responses, and be readily processed into scaffolds with the shape of B-PT-B autografts.
Assuntos
Osso e Ossos/citologia , Técnicas Eletroquímicas/métodos , Ligamentos/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , Peptídeos Catiônicos Antimicrobianos/química , Células da Medula Óssea , Células Cultivadas , Desenho de Equipamento , Feminino , Poliésteres/química , Ratos , Resistência à TraçãoRESUMO
Electrospun meshes suffer from poor cell infiltration and limited thickness, which restrict their use to thin tissue applications. Herein, we demonstrate two complementary processes to overcome these limitations and achieve elastomeric composites that may be suitable for ligament repair. First, C3H10T1/2 mesenchymal stem cells were incorporated into electrospun meshes using a hybrid electrospinning/electrospraying process. Second, electrospun meshes were rolled and formed into composites with an interpenetrating polyethylene glycol (PEG) hydrogel network. Stiffer composites were formed from poly(lactic-co-glycolic acid) (PLGA) meshes, while softer and more elastic composites were formed from poly(ester-urethane urea) (PEUUR) meshes. As-spun PLGA and PEUUR rolled meshes had tensile moduli of 19.2 ± 1.9 and 0.86 ± 0.34 MPa, respectively, which changed to 11.6 ± 4.8 and 1.05 ± 0.39 MPa with the incorporation of a PEG hydrogel phase. In addition, cyclic tensile testing indicated that PEUUR-based composites deformed elastically to at least 10%. Finally, C3H10T1/2 cells incorporated into electrospun meshes survived the addition of the PEG phase and remained viable for up to 5 days. These results indicate that the fabricated cellularized composites are support cyclic mechanical conditioning, and have potential application in ligament repair.
Assuntos
Hidrogel de Polietilenoglicol-Dimetacrilato/química , Ligamentos/química , Células-Tronco Mesenquimais/química , Engenharia Tecidual/métodos , Animais , Linhagem Celular , Hidrogel de Polietilenoglicol-Dimetacrilato/metabolismo , Ácido Láctico/química , Ácido Láctico/metabolismo , Ligamentos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Ácido Poliglicólico/química , Ácido Poliglicólico/metabolismo , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Estresse MecânicoRESUMO
Perfusion bioreactors are a promising in vitro strategy to engineer bone tissue because they supply needed oxygen and nutrients and apply an osteoinductive mechanical stimulus to osteoblasts within large porous three-dimensional scaffolds. Model two-dimensional studies have shown that dynamic flow conditions (e.g., pulsatile oscillatory waveforms) elicit an enhanced mechanotransductive response and elevated expression of osteoblastic proteins relative to steady flow. However, dynamic perfusion of three-dimensional scaffolds has been primarily examined in short term cultures to probe for early markers of mechanotransduction. Therefore, the objective of this study was to investigate the effect of extended dynamic perfusion culture on osteoblastic differentiation of primary mesenchymal stem cells (MSCs). To accomplish this, rat bone marrow-derived MSCs were seeded into porous foam scaffolds and cultured for 15 days in osteogenic medium under pulsatile regimens of 0.083, 0.050, and 0.017 Hz. Concurrently, MSCs seeded in scaffolds were also maintained under static conditions or cultured under steady perfusion. Analysis of the cells after 15 days of culture indicated that alkaline phosphatase (ALP) activity, mRNA expression of osteopontin (OPN), and accumulation of OPN and prostaglandin E(2) were enhanced for all four perfusion conditions relative to static culture. ALP activity, OPN and OC mRNA, and OPN protein accumulation were slightly higher for the intermediate frequency (0.05 Hz) as compared with the other flow conditions, but the differences were not statistically significant. Nevertheless, these results demonstrate that dynamic perfusion of MSCs may be a useful strategy for stimulating osteoblastic differentiation in vitro.
Assuntos
Reatores Biológicos , Diferenciação Celular , Técnicas de Cultura/métodos , Células-Tronco Mesenquimais/citologia , Osteogênese , Fluxo Pulsátil , Fosfatase Alcalina/metabolismo , Animais , Células da Medula Óssea/citologia , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/metabolismo , Dinoprostona/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Ácido Láctico/farmacologia , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/enzimologia , Células-Tronco Mesenquimais/metabolismo , Osteocalcina/genética , Osteogênese/efeitos dos fármacos , Osteopontina/genética , Osteopontina/metabolismo , Ácido Poliglicólico/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Alicerces TeciduaisRESUMO
Polycaprolactone (PCL) fiber scaffolds are attractive, albeit inert, substrates for ligament regeneration, that may be improved by incorporating trophic factors to guide tissue remodeling in vivo. In particular, immobilization of bone morphogenic protein-2 (BMP-2) to the scaffold surface may facilitate rapid and robust integration of the scaffold with adjacent bone tissues. As a first step toward testing this, model PCL surfaces were modified by the addition of heparin (Hep) and BMP-2 to facilitate osteoblastic differentiation. Specifically, Hep was combined with PCL at 0, 0.5, and 1 wt% (denoted as PCL, PCL-0.5Hep, and PCL-1Hep), cast into films, and then BMP-2 was immobilized to surfaces by either adsorption and covalent conjugation. Here, BMP-2 concentration increased systematically with incorporation of Hep, and higher concentrations were achieved by covalent conjugation. Next, blends were electrospun to form thin meshes with fiber diameters of 0.92, 0.62, and 0.54 µm for PCL, PCL-0.5Hep, and PCL-1Hep, respectively. Mesenchymal stem cells (MSCs) had no difficulty attaching to and proliferating on all meshes. Lastly, PCL-1Hep meshes were prepared with adsorbed or covalently conjugated BMP-2 and cultured with MSCs in the absence of osteogenic factors. Under these conditions, alkaline phosphatase activity and deposition of bone sialoprotein, osteopontin, and calcium minerals-markers of osteoblastic differentiation-were significantly higher on surfaces with immobilized BMP-2. Together, these data indicate that covalent immobilization of trophic factors confers bioactivity to scaffolds, which may be applied in a spatially controlled manner for ligament regeneration and bone integration.
Assuntos
Proteína Morfogenética Óssea 2 , Heparina , Células-Tronco Mesenquimais/fisiologia , Poliésteres , Alicerces Teciduais , Animais , Diferenciação Celular , Masculino , Osteoblastos/fisiologia , Osteogênese , Ratos Sprague-Dawley , Engenharia TecidualRESUMO
Amorphous calcium phosphates (ACPs) are attractive fillers for osseous defects and are stabilized through the incorporation of transition metals such as zirconium and zinc. As ACP converts in solution to hydroxyapatite (HAP) in a manner marked by a transient release of calcium and phosphate ions, it is capable of stimulating osteoblastic differentiation. Zinc is known to retard ACP conversion to HAP, and--when incorporated into ceramic biomaterials--has been shown to stimulate osteoblastic differentiation. Because zinc deficiency in vivo is marked by skeletal defects, we postulated that zinc ions released from ACP and other minerals could stimulate proliferation and osteoblastic differentiation of progenitor cells. To test this hypothesis, rat bone marrow stromal cells were cultured in osteogenic medium containing basal (3 x 10(-6) M) or supplemented Zn(2+) concentrations (1 x 10(-5) and 4 x 10(-5) M) for up to 3 weeks. No significant effects of zinc concentration on cell number, alkaline phosphatase activity, total protein content, collagen synthesis, or matrix mineralization were found.
Assuntos
Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Zinco/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Calcificação Fisiológica/efeitos dos fármacos , Contagem de Células , Células Cultivadas , Colágeno/biossíntese , Masculino , Ratos , Ratos Sprague-Dawley , Solubilidade/efeitos dos fármacos , Células Estromais/citologia , Células Estromais/efeitos dos fármacosRESUMO
Segmented polyurethanes have been used extensively in implantable medical devices, but their tunable mechanical properties make them attractive for examining the effect of biomaterial modulus on engineered musculoskeletal tissue development. In this study, a family of segmented degradable poly(esterurethane urea)s (PEUURs) were synthesized from 1,4-diisocyanatobutane, a poly(epsilon-caprolactone) (PCL) macrodiol soft segment and a tyramine-1,4-diisocyanatobutane-tyramine chain extender. By systematically increasing the PCL macrodiol molecular weight from 1100 to 2700Da, the storage modulus, crystallinity and melting point of the PCL segment were systematically varied. In particular, the melting temperature, T(m), increased from 21 to 61 degrees C and the storage modulus at 37 degrees C increased from 52 to 278MPa with increasing PCL macrodiol molecular weight, suggesting that the crystallinity of the PCL macrodiol contributed significantly to the mechanical properties of the polymers. Bone marrow stromal cells were cultured on rigid polymer films under osteogenic conditions for up to 21 days. Cell density, alkaline phosphatase activity, and osteopontin and osteocalcin expression were similar among PEUURs and comparable to poly(d,l-lactic-coglycolic acid). This study demonstrates the suitability of this family of PEUURs for tissue engineering applications, and establishes a foundation for determining the effect of biomaterial modulus on bone tissue development.
Assuntos
Materiais Biocompatíveis/química , Elastômeros/química , Poliuretanos/química , Engenharia Tecidual/métodos , Ureia/química , Fosfatase Alcalina/metabolismo , Animais , Materiais Biocompatíveis/síntese química , Osso e Ossos/citologia , Contagem de Células , Técnicas de Cultura de Células , Células Cultivadas , Elastômeros/síntese química , Masculino , Estrutura Molecular , Osteocalcina/metabolismo , Osteopontina/metabolismo , Poliuretanos/síntese química , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Células Estromais/citologia , TemperaturaRESUMO
Engineered ligament tissues are promising materials for the repair of tears and ruptures, but require the development of biomaterial scaffolds that not only support physiologically relevant loads, but also possess architectures capable of orienting cell adhesion and extracellular matrix deposition. Based on evidence that micron-scale topographic features induce cell orientation through a contact guidance phenomenon, we postulate that oriented micron-scale fiber meshes-formed by the electrospinning process-can regulate cell morphology. To test this, fused fiber meshes of poly(d,l-lactic-co-glycolic acid) (PLGA) were electrospun onto rigid supports under conditions that produced mean fiber diameters of 0.14-3.6 microm, and angular standard deviations of 31-60 degrees . Analysis of the morphology of adherent NIH 3T3 fibroblasts indicated that projected cell area and aspect ratio increased systematically with both increasing fiber diameter and degree of fiber orientation. Importantly, cell morphology on 3.6 microm fibers was similar to that on spincoated PLGA films. Finally, cell densities on electrospun meshes were not significantly different from spincoated PLGA, indicating that cell proliferation is not sensitive to fiber diameter or orientation.
Assuntos
Proliferação de Células , Fibroblastos , Ácido Láctico/química , Ácido Poliglicólico/química , Polímeros/química , Animais , Materiais Biocompatíveis/metabolismo , Técnicas de Cultura de Células , Forma Celular , Fibroblastos/citologia , Fibroblastos/fisiologia , Teste de Materiais , Camundongos , Microscopia Eletrônica de Varredura , Células NIH 3T3 , Tamanho da Partícula , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Engenharia TecidualRESUMO
Electrospinning is a promising method to construct fused-fiber biomaterial scaffolds for tissue engineering applications, but the efficacy of this approach depends on how substrate topography affects cell function. Previously, it has been shown that linear, parallel raised features with length scales of 0.5-2 microm direct cell orientation through the phenomenon of contact guidance, and enhance phenotypic markers of osteoblastic differentiation. To determine how the linear, random raised features produced by electrospinning affect proliferation and differentiation of osteoprogenitor cells, poly(lactic acid) and poly(ethylene glycol)-poly(lactic acid) diblock copolymers were electrospun with mean fiber diameters of 0.14-2.1 microm onto rigid supports. MC3T3-E1 osteoprogenitor cells cultured on fiber surfaces in the absence of osteogenic factors exhibited a lower cell density after 7 and 14 days of culture than cells cultured on spin-coated surfaces, but cell density increased with fiber diameter. However, in the presence of osteogenic factors (2 mM beta-glycerophosphate, 0.13 mM L-ascorbate-2-phosphate), cell density after 7 and 14 days of culture on fiber surfaces was comparable to or exceeded spin-coated controls, and alkaline phosphatase activity after 14 days was comparable. Examination of cell morphology revealed that cells grown on fibers had smaller projected areas than those on planar surfaces. However, cells attached to electrospun substrates of 2.1 microm diameter fibers exhibited a higher cell aspect ratio than cells on smooth surfaces. These studies show that topographical factors designed into biomaterial scaffolds can regulate spreading, orientation, and proliferation of osteoblastic cells.
Assuntos
Substitutos Ósseos/química , Técnicas de Cultura de Células/métodos , Eletroquímica/métodos , Ácido Láctico/química , Osteoblastos/citologia , Osteoblastos/fisiologia , Polímeros/química , Engenharia Tecidual/métodos , Células 3T3 , Animais , Materiais Biocompatíveis/química , Substitutos Ósseos/análise , Diferenciação Celular , Movimento Celular/fisiologia , Proliferação de Células , Ácido Láctico/análise , Teste de Materiais , Camundongos , Conformação Molecular , Peso Molecular , Tamanho da Partícula , Poliésteres , Polímeros/análise , Rotação , Propriedades de Superfície , Têxteis/análiseRESUMO
Calcium phosphate bioceramics, such as hydroxyapatite, have long been used as bone substitutes because of their proven biocompatibility and bone binding properties in vivo. Recently, a zirconia-hybridized pyrophosphate-stabilized amorphous calcium phosphate (Zr-ACP) has been synthesized, which is more soluble than hydroxyapatite and allows for controlled release of calcium and phosphate ions. These ions have been postulated to increase osteoblast differentiation and mineralization in vitro. The focus of this work is to elucidate the physicochemical properties of Zr-ACP and to measure cell response to Zr-ACP in vitro using a MC3T3-E1 mouse calvarial-derived osteoprogenitor cell line. Cells were cultured in osteogenic medium and mineral was added to culture at different stages in cell maturation. Culture in the presence of Zr-ACP showed significant increases in cell proliferation, alkaline phosphatase activity (ALP), and osteopontin (OPN) synthesis, whereas collagen synthesis was unaffected. In addition, calcium and phosphate ion concentrations and medium pH were found to transiently increase with the addition of Zr-ACP, and are hypothesized to be responsible for the osteogenic effect of Zr-ACP.
Assuntos
Substitutos Ósseos , Pirofosfato de Cálcio , Osteoblastos/fisiologia , Osteogênese/fisiologia , Crânio/fisiologia , Zircônio , Animais , Fosfatos de Cálcio , Pirofosfato de Cálcio/química , Diferenciação Celular/fisiologia , Linhagem Celular , Proliferação de Células , Teste de Materiais , Camundongos , Osteoblastos/citologia , Crânio/citologia , Zircônio/químicaRESUMO
Electrospun microfibers are attractive for the engineering of oriented tissues because they present instructive topographic and mechanical cues to cells. However, high-density microfiber networks are too cell-impermeable for most tissue applications. Alternatively, the distribution of sparse microfibers within a three-dimensional hydrogel could present instructive cues to guide cell organization while not inhibiting cell behavior. In this study, thin (â¼5 fibers thick) layers of aligned microfibers (0.7 µm) were embedded within collagen hydrogels containing mesenchymal stem cells (MSCs), cultured for up to 14 days, and assayed for expression of ligament markers and imaged for cell organization. These microfibers were generated through the electrospinning of polycaprolactone (PCL), poly(ester-urethane) (PEUR), or a 75/25 PEUR/PCL blend to produce microfiber networks with elastic moduli of 31, 15, and 5.6 MPa, respectively. MSCs in composites containing 5.6 MPa fibers exhibited increased expression of the ligament marker scleraxis and the contractile phenotype marker α-smooth muscle actin versus the stiffer fiber composites. Additionally, cells within the 5.6 MPa microfiber composites were more oriented compared to cells within the 15 and 31 MPa microfiber composites. Together, these data indicate that the mechanical properties of microfiber/collagen composites can be tuned for the engineering of ligament and other target tissues. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1894-1901, 2016.
Assuntos
Colágeno/farmacologia , Módulo de Elasticidade , Ligamentos/fisiologia , Células-Tronco Mesenquimais/citologia , Engenharia Tecidual/métodos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Forma Celular/efeitos dos fármacos , Colágeno/genética , Colágeno/metabolismo , DNA/análise , Ligamentos/efeitos dos fármacos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Poliésteres/química , Poliésteres/farmacologia , Poliuretanos/química , Poliuretanos/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Estresse MecânicoRESUMO
This work investigates the potential of cell layer-electrospun mesh constructs as coronary artery bypass grafts. These cell-mesh constructs were generated by first culturing a confluent layer of 10T½ smooth muscle progenitor cells on a high strength electrospun mesh with uniaxially aligned fibers. Cell-laden mesh sheets were then wrapped around a cylindrical mandrel such that the mesh fibers were aligned circumferentially. The resulting multi-layered constructs were then cultured for 4 wks in media supplemented with TGF-ß1 and ascorbic acid to support 10T½ differentiation toward a smooth muscle cell-like fate as well as to support elastin and collagen production. The underlying hypothesis of this work was that extracellular matrix (ECM) deposited by the cell layers would act as an adhesive agent between the individual mesh layers, providing strength to the construct as well as a source for structural elasticity at low strains. In addition, the structural anisotropy of the mesh would inherently guide desired circumferential cell and ECM alignment. Results demonstrate that the cell-mesh constructs exhibited a J-shaped circumferential stress-strain response similar to that of native coronary artery, while also displaying acceptable tensile strength. Furthermore, associated 10T½ cells and deposited collagen fibers showed a high degree of circumferential alignment. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2200-2209, 2016.
Assuntos
Prótese Vascular , Ponte de Artéria Coronária , Vasos Coronários , Matriz Extracelular/metabolismo , Miócitos de Músculo Liso/metabolismo , Poliuretanos/química , Animais , Linhagem Celular , CamundongosRESUMO
Bone marrow stromal cells (BMSCs) are multipotent progenitor cells with a capacity to form bone tissue in vivo, and to differentiate into the osteoblastic lineage in vitro. Drawing on evidence that bone is mechanosensitive and mechanical stimuli are anabolic, we postulate that proliferation and osteoblastic differentiation of BMSCs may be stimulated by mechanical forces. In this study, BMSCs cultured in the presence of osteogenic factors (dexamethasone, beta-glycerophosphate, and ascorbate) were stimulated repeatedly (every second day) with shearing flow (1.6 dyn/cm(2)) for 5, 30, or 120 min, and assayed for systematic changes in cell number and phenotypic markers of osteoblastic differentiation. Cells exposed to fluid flow on days 2 and 4 after the addition of osteogenic factors and assayed at day 6 exhibited a modest decrease in cell number and increase in normalized alkaline phosphatase activity, suggesting the detachment of a non-osteogenic subpopulation. Cells exposed to fluid flow on days 6, 8, 10, and 12 and assayed at day 20 demonstrated maximal expression of osteopontin and bone sialoprotein mRNA with 30 min duration of flow. Concurrently, at day 20 expression of the adipogenic marker, lipoprotein lipase, was minimal with a 120-min duration of flow. These results indicate that repeated application of shear stress stimulates late phenotypic markers of osteoblastic differentiation of BMSCs in a manner that depends on the duration of stimulus. Finally, accumulation of prostaglandin E(2) in culture medium in response to shearing flow systematically decreased with repeated exposure to 30 and 120 min of shear stress (from day 6 to day 12), suggesting an adaptation of the cells to fluid flow.
Assuntos
Células da Medula Óssea/fisiologia , Sialoglicoproteínas/genética , Células Estromais/fisiologia , Fosfatase Alcalina/metabolismo , Animais , Ácido Ascórbico/farmacologia , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Contagem de Células , Células Cultivadas , Dexametasona/farmacologia , Dinoprostona/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glicerofosfatos/farmacologia , Sialoproteína de Ligação à Integrina , Lipase Lipoproteica/genética , Microfluídica , Osteopontina , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Sialoglicoproteínas/metabolismo , Células Estromais/citologia , Células Estromais/metabolismo , Fatores de TempoRESUMO
Electrostatic layer-by-layer film assembly is an attractive way to non-covalently incorporate proteins and bioactive moieties into the surface of conventional biomaterials. Selection of polycationic and polyanionic components and deposition conditions can be used to control the interfacial properties, and through them protein adsorption, cell adhesion, and tissue development. In this study the polycation was poly(allylamine hydrochloride) (PAH), which is a weak base and consequently adsorbs at interfaces in a pH-dependent manner, and the polyanion was heparin, which is capable of interacting with many adhesion ligands and growth factors. PAH/heparin multilayer films were formed using PAH solutions of pH 6.4, 7.4, 8.4, and 9.4. Film thickness increased both with the number of PAH/heparin bilayers and the pH of the PAH solution. Films consisting of 10 bilayers with heparin topmost exhibited similar bulk atomic compositions and penetration of PAH into the heparin top layer. Finally, fibronectin adsorption and cell adhesion were maximal at an intermediate pH (pH 8.4>pH 9.4>pH 7.4). These results demonstrate that heparin-containing electrostatic films support cell adhesion and protein adsorption in a manner sensitive to film deposition conditions.
Assuntos
Adsorção , Materiais Biocompatíveis/química , Heparina/química , Poliaminas/química , Animais , Ânions , Cátions , Adesão Celular , Fibronectinas/química , Fibronectinas/metabolismo , Concentração de Íons de Hidrogênio , Bicamadas Lipídicas , Camundongos , Modelos Químicos , Osteoblastos/metabolismo , Polieletrólitos , Espectrometria por Raios X , Eletricidade Estática , Propriedades de SuperfícieRESUMO
Many polyurethane elastomers display excellent mechanical properties and adequate biocompatibility. However, many medical-grade polyurethanes are prepared from aromatic diisocyanates and can degrade in vivo to carcinogenic aromatic diamines, although the question of whether the concentrations of these harmful degradation products attain physiologically relevant levels is currently unresolved and strongly debated. It is therefore desirable to synthesize new medical-grade polyurethanes from less toxic aliphatic diisocyanates. In this paper, biocompatible segmented polyurethane elastomers were synthesized from aliphatic diisocyanates (1,4-diisocyanatobutane (BDI) and lysine methyl ester diisocyanate (LDI)), novel diurea diol chain extenders based on tyrosine and tyramine, and a model poly(ethylene glycol) (PEG) diol soft segment. The objectives were to design a hard segment similar in structure to that of MDI-based polyurethanes and also investigate the effects of systematic changes in structure on mechanical and biological properties. The non-branched, symmetric polyurethane prepared from BDI and a tyramine-based chain extender had the highest modulus at 37 degrees C. Introduction of symmetric short-chain branches (SCBs) incorporated in the tyrosine-based chain extender lowered the modulus by an order of magnitude. Polyurethanes prepared from LDI were soft polymers that had a still lower modulus due to the asymmetric SCBs that hindered hard segment packing. Polyurethanes prepared from tyramine and tyrosine chain extenders thermally degraded at temperatures ranging from 110 to 150 degrees C, which are lower than that reported previously for phenyl urethanes. All four polyurethanes supported the attachment, proliferation, and high viability of MG-63 human osteoblast-like cells in vitro. Therefore, the non-cytotoxic chemistry of these polyurethanes make them good candidates for further development as biomedical implants.
Assuntos
Materiais Biocompatíveis/química , Isocianatos/química , Osteossarcoma/patologia , Osteossarcoma/fisiopatologia , Poliuretanos/química , Engenharia Tecidual/métodos , Ureia/química , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Reagentes de Ligações Cruzadas/química , HumanosRESUMO
Bone marrow stromal cells (BMSCs) are a promising component for engineered bone tissues, but in vitro formation of a bonelike tissue requires culture conditions that direct these multipotent cells toward osteoblastic maturation. Fluid flow has been postulated to stimulate bone tissue development in vivo, but the effect of shear stress on proliferation and differentiation of osteoprogenitor cell cultures in vitro has not been examined closely. In this study BMSCs were cultured on fibronectin-coated substrates and exposed intermittently (for 30 min 3, 5, 7, 9, 11, and 13 days after seeding) to a spatially dependent range of shear stresses (0.36 to 2.7 dyn/cm(2)) using a radial-flow chamber. After 7 days cell density did not vary between sheared and control cell layers. In contrast, after 21 days the accumulation of osteocalcin protein (OC) in cell layers was increased significantly relative to static controls, while the quantity of multilayer cell aggregates (i.e., bone nodules) was diminished. Neither of these effects varied systematically with shear magnitude. Finally, pretreatment of cultures with the cyclooxygenase (COX)-2-specific inhibitor NS-398 blocked prostaglandin secretion in response to shearing flow and significantly reduced OC accumulation in cell layers. These results provide evidence that flow stimulates osteoblastic maturation but not proliferation of bone marrow stromal cells and that prostaglandin signaling is involved in this effect.
Assuntos
Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Mecanotransdução Celular/fisiologia , Osteoblastos/citologia , Osteoblastos/fisiologia , Osteocalcina/biossíntese , Engenharia Tecidual/métodos , Animais , Células da Medula Óssea/efeitos dos fármacos , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Inibidores de Ciclo-Oxigenase/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Masculino , Mecanotransdução Celular/efeitos dos fármacos , Microfluídica/métodos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/efeitos dos fármacos , Células-Tronco Multipotentes/fisiologia , Nitrobenzenos/farmacologia , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Ratos , Ratos Sprague-Dawley , Resistência ao Cisalhamento , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/fisiologia , Sulfonamidas/farmacologiaRESUMO
Synthetic biomaterials intended for the reconstruction of tissues and organs must be capable of sustaining adhesive contact with adjacent cells and tissues under mechanical and hydrodynamic stresses. To facilitate this adhesion, extracellular matrix proteins or peptide sequences are frequently immobilized to the biomaterial interface. These ligands enhance cell attachment by raising the number of cell receptor/ligand interactions, but consequently they may alter the mechanism of cell detachment. In particular, as the cell membrane is more strongly immobilized to the substratum, the tendency for cell detachment to involve membrane rupture may increase. To test this hypothesis, cells were fluorescent stained with a membrane dye, allowed to attach to fibronectin-coated model substrates for 30 min, and then subjected to a spatially dependent range of shear stress for 5 min (28-220 dyn/cm2) using a radial-flow chamber. Phase-contrast and fluorescent images were analyzed to determine the probability for cell detachment and the area of fluorescent debris left by detaching cells as a function of fibronectin concentration, magnitude of shear stress, and time. It was found at all concentrations of fibronectin that the majority of detaching cells left membrane fragments, the mean size of these fragments was independent of shear stress, and the shape independent of the direction of flow. However, mean fragment area increased with concentration of fibronectin and decreased with duration of shearing flow. We postulate that the area of debris reflects the extent of cell attachment prior to the application of shear and that adhesive complexes can disassemble at the onset of flow.
Assuntos
Adesão Celular/fisiologia , Membrana Celular/fisiologia , Materiais Revestidos Biocompatíveis , Fibronectinas , Células 3T3 , Animais , CamundongosRESUMO
Efforts to develop engineered tendons and ligaments have focused on the use of a biomaterial scaffold and a stem cell source. However, the ideal scaffold microenvironment to promote stem cell differentiation and development of organized extracellular matrix is unknown. Through electrospinning, fibre scaffolds can be designed with tailorable architectures to mimic the intended tissue. In this study, the effects of fibre diameter and orientation were examined by electrospinning thin mats, consisting of small (< 1 µm), medium (1-2 µm) or large (> 2 µm) diameter fibres with either random or aligned fibre orientation. C3H10T1/2 model stem cells were cultured on the six different electrospun mats, as well as smooth spin-coated films, and the morphology, growth and expression of tendon/ligament genes were evaluated. The results demonstrated that fibre diameter affects cellular behaviour more significantly than fibre alignment. Initially, cell density was greater on the small fibre diameter mats, but similar cell densities were found on all mats after an additional week in culture. After 2 weeks, gene expression of collagen 1α1 and decorin was increased on all mats compared to films. Expression of the tendon/ligament transcription factor scleraxis was suppressed on all electrospun mats relative to spin-coated films, but expression on the large-diameter fibre mats was consistently greater than on the medium-diameter fibre mats. These results suggest that larger-diameter fibres (e.g. > 2 µm) may be more suitable for in vitro development of a tendon/ligament tissue.
Assuntos
Diferenciação Celular , Ligamentos/citologia , Células-Tronco Mesenquimais/citologia , Tendões/citologia , Animais , Sequência de Bases , Primers do DNA , Camundongos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Calcium phosphate ceramics (CPCs) have been widely used as biomaterials for the regeneration of bone tissue because of their ability to induce osteoblastic differentiation in progenitor cells. Despite the progress made towards fabricating CPCs possessing a range of surface features and chemistries, the influence of material properties in orchestrating cellular events such as adhesion and differentiation is still poorly understood. Specifically, questions such as why certain CPCs may be more osteoinductive than others, and how material properties contribute to osteoinductivity/osteoconductivity remain unanswered. Therefore, this review article systematically discusses the effects of the physical (e.g. surface roughness) and chemical properties (e.g. solubility) of CPCs on protein adsorption, cell adhesion and osteoblastic differentiation in vitro. The review also provides a summary of possible signaling pathways involved in osteoblastic differentiation in the presence of CPCs. In summary, these insights on the contribution of material properties towards osteoinductivity and the role of signaling molecules involved in osteoblastic differentiation can potentially aid the design of CPC-based biomaterials that support bone regeneration without the need for additional biochemical supplements.
Assuntos
Substitutos Ósseos/química , Fosfatos de Cálcio/química , Cerâmica/química , Osteoblastos/citologia , Osteoblastos/fisiologia , Osteogênese/fisiologia , Engenharia Tecidual/instrumentação , Animais , Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células , Sobrevivência Celular/fisiologia , Desenho de Equipamento , Humanos , Propriedades de SuperfícieRESUMO
Segmented polyurethanes (PURs), consisting of degradable poly(a-hydroxy ester) soft segments and aminoacid-derived chain extenders, are biocompatible elastomers with tunable mechanical and degradative properties suitable for a variety of tissue-engineering applications. In this study, a family of linear PURs synthesized from poly(ϵ-caprolactone) (PCL) diol, 1,4-diisocyanobutane and tyramine with theoretical PCL contents of 65-80 wt% were processed into porous foam scaffolds and evaluated for their ability to support osteoblastic differentiation in vitro. Differential scanning calorimetry and mechanical testing of the foams indicated increasing polymer crystallinity and compressive modulus with increasing PCL content. Next, bone marrow stromal cells (BMSCs) were seeded into PUR scaffolds, as well as poly(lactic-co-glycolic acid) (PLGA) scaffolds, and maintained under osteogenic conditions for 14 and 21 days. Analysis of cell number indicated a systematic decrease in cell density with increasing PUR stiffness at both 14 and 21 days in culture. However, at these same time points the relative mRNA expression for the bone-specific proteins osteocalcin and the growth factors bone morphogenetic protein-2 and vascular endothelial growth factor gene expression were similar among the PURs. Finally, prostaglandin E2 production, alkaline phosphatase activity and osteopontin mRNA expression were highly elevated on the most-crystalline PUR scaffold as compared to the PLGA and PUR scaffolds. These results suggest that both the modulus and crystallinity of the PUR scaffolds influence cell proliferation and the expression of osteoblastic proteins.