RESUMO
Engineering the meniscus is challenging due to its bizonal structure; the tissue is cartilaginous at the inner portion and fibrous at the outer portion. Here, we constructed an artificial meniscus mimicking the biochemical organization of the native tissue by 3D printing a meniscus shaped PCL scaffold and then impregnating it with agarose (Ag) and gelatin methacrylate (GelMA) hydrogels in the inner and outer regions, respectively. After incubating the constructs loaded with porcine fibrochondrocytes for 8 weeks, we demonstrated that presence of Ag enhanced glycosaminoglycan (GAG) production by about 4 fold (p < 0.001), while GelMA enhanced collagen production by about 50 fold (p < 0.001). In order to mimic the physiological loading environment, meniscus shaped PCL/hydrogel constructs were dynamically stimulated at strain levels gradually increasing from the outer region (2% of initial thickness) towards the inner region (10%). Incorporation of hydrogels protected the cells from the mechanical damage caused by dynamic stress. Dynamic stimulation resulted in increased ratio of collagen type II (COL 2) in the Ag-impregnated inner region (from 50% to 60% of total collagen), and increased ratio of collagen type I (COL 1) in the GelMA-impregnated outer region (from 60% to 70%). We were able to engineer a meniscus, which is cartilage-like at the inner portion and fibrocartilage-like at the outer portion. Our construct has a potential for use as a substitute for total meniscus replacement.
Assuntos
Materiais Biomiméticos/química , Hidrogéis/química , Menisco/metabolismo , Poliésteres/química , Impressão Tridimensional , Animais , Forma Celular , Sobrevivência Celular , Colágeno/metabolismo , Feminino , Glicosaminoglicanos/metabolismo , Sulfatos/metabolismo , Suínos , Alicerces Teciduais/químicaRESUMO
In this study, porcine fibrochondrocyte-seeded agarose, methacrylated gelatin (GelMA), methacrylated hyaluronic acid (MeHA) and GelMA-MeHA blend hydrogels, and 3D printed PCL scaffolds were tested under dynamic compression for potential meniscal regeneration in vitro. Cell-carrying hydrogels produced higher levels of extracellular matrix (ECM) components after a 35-day incubation than the 3D printed PCL. Cells on GelMA exhibited strong cell adhesion (evidenced with intense paxillin staining) and dendritic cell morphology, and produced an order of magnitude higher level of collagen (pâ¯<â¯0.05) than other materials. On the other hand, cells in agarose exhibited low cell adhesion and round cell morphology, and produced higher levels of glycosaminoglycans (GAGs) (pâ¯<â¯0.05) than other materials. A low level of ECM production and a high level of cell proliferation were observed on the 3D printed PCL. Dynamic compression at 10% strain enhanced GAG production in agarose (pâ¯<â¯0.05), and collagen production in GelMA. These results show that hydrogels have a higher potential for meniscal regeneration than the 3D printed PCL, and depending on the material used, fibrochondrocytes could be directed to proliferate or produce cartilaginous or fibrocartilaginous ECM. Agarose and MeHA could be used for the regeneration of the inner region of meniscus, while GelMA for the outer region.
Assuntos
Gelatina/química , Ácido Hialurônico/química , Hidrogéis/química , Menisco/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Sefarose/química , Sefarose/farmacologia , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Fenômenos Mecânicos , Menisco/fisiologia , Poliésteres/farmacologia , Impressão Tridimensional , Suínos , Alicerces TeciduaisRESUMO
Growth factors have become an important component for tissue engineering and regenerative medicine. Insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-beta 1) in particular have great significance in cartilage tissue engineering. Here, we describe sequential release of IGF-I and TGF-beta 1 from modular designed poly(l,d-lactic-co-glycolic acid) (PLGA) scaffolds. Growth factors were encapsulated in PLGA microspheres using spontaneous emulsion, and in vitro release kinetics was characterized by ELISA. Incorporating BSA in the IGF-I formulations decreased the initial burst from 80% to 20%, while using uncapped PLGA rather than capped decreased the initial burst of TGF-beta 1 from 60% to 0% upon hydration. The bioactivity of released IGF-I and TGF-beta 1 was determined using MCF-7 proliferation assay and HT-2 inhibition assay, respectively. Both growth factors were released for up to 70 days in bioactive form. Scaffolds were fabricated by fusing bioactive IGF-I and TGF-beta 1 microspheres with dichloromethane vapor. Three scaffolds with tailored release kinetics were fabricated: IGF-I and TGF-beta 1 released continuously, TGF-beta 1 with IGF-I released sequentially after 10 days, and IGF-I with TGF-beta 1 released sequentially after 7 days. Scaffold swelling and degradation were characterized, indicating a peak swelling ratio of 4 after 7 days of incubation and showing 50% mass loss after 28 days, both consistent with scaffold release kinetics. The ability of these scaffolds to release IGF-I and TGF-beta 1 sequentially makes them very useful for cartilage tissue engineering applications.
Assuntos
Fator de Crescimento Insulin-Like I/química , Ácido Láctico/química , Microesferas , Ácido Poliglicólico/química , Fator de Crescimento Transformador beta1/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia em Gel , Portadores de Fármacos , Ensaio de Imunoadsorção Enzimática , Humanos , Fator de Crescimento Insulin-Like I/farmacocinética , Fator de Crescimento Insulin-Like I/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Fator de Crescimento Transformador beta1/farmacocinética , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Decellularization of cartilage enables the use of cartilage allografts or xenografts as natural scaffolds for repair and regeneration of injured cartilage. The preservation of the extracellular matrix ultrastructure of the graft makes this a promising tool for cartilage tissue engineering. We have optimized the decellularization protocol by enzymatically digesting proteoglycans while preserving the native collagen architecture. Here we describe our methods for cartilage decellularization and cell labeling for the tracking of infiltration for recellularization in detail.
Assuntos
Cartilagem Articular/química , Cartilagem Articular/citologia , Matriz Extracelular/química , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Cartilagem Articular/ultraestrutura , Rastreamento de Células/métodos , Matriz Extracelular/ultraestrutura , Células-Tronco Mesenquimais/citologia , SuínosRESUMO
The meniscus is the most commonly injured structure in the human knee. Meniscus deficiency has been shown to lead to advanced osteoarthritis (OA) due to abnormal mechanical forces, and replacement strategies for this structure have lagged behind other tissue engineering endeavors. The challenges include the complex 3D structure with individualized size parameters, the significant compressive, tensile and shear loads encountered, and the poor blood supply. In this progress report, a review of the current clinical treatments for different types of meniscal injury is provided. The state-of-the-art research in cellular therapies and novel cell sources for these therapies is discussed. The clinically available cell-free biomaterial implants and the current progress on cell-free biomaterial implants are reviewed. Cell-based tissue engineering strategies for the repair and replacement of meniscus are presented, and the current challenges are identified. Tissue-engineered meniscal biocomposite implants may provide an alternative solution for the treatment of meniscal injury to prevent OA in the long run, because of the limitations of the existing therapies.
Assuntos
Bioprótese , Menisco , Regeneração , Lesões do Menisco Tibial/terapia , Engenharia Tecidual/métodos , Animais , Humanos , Menisco/lesões , Menisco/fisiologia , Osteoartrite do Joelho/prevenção & controleRESUMO
Articular cartilage has a limited capacity to heal itself and thus focal defects often result in the development of osteoarthritis. Current cartilage tissue engineering strategies seek to regenerate injured tissue by creating scaffolds that aim to mimic the unique structure and composition of native articular cartilage. Decellularization is a novel strategy that aims to preserve the bioactive factors and 3D biophysical environment of the native extracellular matrix while removing potentially immunogenic factors. The purpose of this study was to develop a procedure that can enable decellularization and recellularization of intact articular cartilage matrix. Full-thickness porcine articular cartilage plugs were decellularized with a series of freeze-thaw cycles and 0.1% (w/v) sodium dodecyl sulfate detergent cycles. Chondroitinase ABC (ChABC) was applied before the detergent cycles to digest glycosaminoglycans in order to enhance donor chondrocyte removal and seeded cell migration. Porcine synovium-derived mesenchymal stem cells were seeded onto the decellularized cartilage scaffolds and cultured for up to 28 days. The optimized decellularization protocol removed 94% of native DNA per sample wet weight, while collagen content and alignment were preserved. Glycosaminoglycan depletion prior to the detergent cycles increased removal of nuclear material. Seeded cells infiltrated up to 100 µm into the cartilage deep zone after 28 days in culture. ChABC treatment enhances decellularization of the relatively dense, impermeable articular cartilage by reducing glycosaminoglycan content. ChABC treatment did not appear to affect cell migration during recellularization under static, in vitro culture, highlighting the need for more dynamic seeding methods.
Assuntos
Cartilagem Articular/química , Condroitina ABC Liase/química , Matriz Extracelular/química , Células-Tronco Mesenquimais/citologia , Proteoglicanas/química , Membrana Sinovial/citologia , Alicerces Teciduais/química , Animais , Técnicas de Cultura de Células , Movimento Celular , Células Cultivadas , Células-Tronco Mesenquimais/metabolismo , Suínos , Membrana Sinovial/metabolismoRESUMO
BACKGROUND: Although patients with an anterior cruciate ligament (ACL) injury have a high risk of developing posttraumatic osteoarthritis (PTOA), the role of meniscus hypertrophy and mineralization in PTOA after an ACL injury remains unknown. PURPOSE/HYPOTHESIS: The purpose of this study was to determine if menisci respond to abnormal loading and if an ACL injury results in meniscus hypertrophy and calcification. The hypotheses were that (1) abnormal mechanical loading after an ACL injury induces meniscus hypertrophy and mineralization, which correlates to articular cartilage damage in vivo, and (2) abnormal mechanical loading on bovine meniscus explants induces the overexpression of hypertrophic and mineralization markers in vitro. STUDY DESIGN: Controlled laboratory study. METHODS: In vivo guinea pig study (hypothesis 1): Three-month-old male Hartley guinea pigs (n = 9) underwent ACL transection (ACLT) on the right knee; the left knee served as the control. Calcification in the menisci was evaluated by calcein labeling 1 and 5 days before knee harvesting at 5.5 months. Cartilage and meniscus damage and mineralization were quantified by the Osteoarthritis Research Society International score and meniscus grade, respectively. Indian hedgehog (Ihh), matrix metalloproteinase-13 (MMP-13), collagen type X (Col X), progressive ankylosis homolog (ANKH), ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1), alkaline phosphatase (ALP), inorganic pyrophosphate (PPi), and inorganic phosphate (Pi) concentrations were evaluated by immunohistochemistry and enzyme-linked immunosorbent assay. In vitro bovine meniscus explant study (hypothesis 2): Bovine meniscus explants were subjected to 25% strain at 0.3 Hz for 1, 2, and 3 hours. Cell viability was determined using live/dead staining. The levels of mRNA expression and protein levels were measured using real-time quantitative reverse transcription polymerase chain reaction and Western blot after 24, 48, and 72 hours in culture. The conditioned medium was collected for sulfated glycosaminoglycan (GAG) release and Pi/PPi assay. RESULTS: In vivo guinea pig study: Meniscus size and area as well as intensity of meniscus calcification were significantly increased in the ACLT group compared with the control group. Both calcified area and intensity were correlated with cartilage damage in the ACLT group (meniscus calcified area: r = 0.925, P < .0001; meniscus calcified intensity: r = 0.944, P < .0001). Ihh, MMP-13, Col X, ANKH, ENPP1, and ALP expression were increased in the ACLT group compared with the control group. The Pi level and Pi/PPi ratio increased by 63% and 42%, respectively, in the ACLT group compared with the control group. In vitro bovine meniscus explant study: Cell death was found in the superficial zone of the bovine meniscus explants after loading for 3 hours. The mRNA expression and protein levels of MMP-13, ANKH, ENPP1, and ALP were up-regulated in all 3-hour loaded samples. The Pi/PPi ratio and sulfated GAG content in the culture medium were increased in the 3-hour loaded group. CONCLUSION: Meniscus hypertrophy and mineralization correlated to cartilage degeneration after ACL injuries. CLINICAL RELEVANCE: The study data suggest that the suppression of meniscus hypertrophy and calcification may decrease the risk of PTOA after ACL injuries.
Assuntos
Ligamento Cruzado Anterior/patologia , Calcinose/patologia , Meniscos Tibiais/patologia , Animais , Ligamento Cruzado Anterior/metabolismo , Lesões do Ligamento Cruzado Anterior , Calcinose/metabolismo , Cartilagem Articular/lesões , Feminino , Cobaias , Hipertrofia , Traumatismos do Joelho , Masculino , Metaloproteinase 13 da Matriz/metabolismo , Lesões do Menisco TibialRESUMO
Hydrodynamic forces in bioreactors can decisively influence extracellular matrix deposition in engineered cartilage constructs. In the present study, the reduced fluid shear, high-axial mixing environment provided by a wavy-walled bioreactor was exploited in the cultivation of cartilage constructs using polyglycolic acid scaffolds seeded with bovine articular chondrocytes. Increased growth as defined by weight, cell proliferation and extracellular matrix deposition was observed in cartilage constructs from wavy-walled bioreactors in comparison with those from spinner flasks cultured under the same conditions. The wet weight composition of 4-week constructs from the wavy-walled bioreactor was similar to that of spinner flask constructs, but the former were 60% heavier due to equally higher incorporation of extracellular matrix and 30% higher cell population. It is most likely that increased construct matrix incorporation was a result of increased mitotic activity of chondrocytes cultured in the environment of the wavy-walled bioreactor. A layer of elongated cells embedded in type I collagen formed at the periphery of wavy-walled bioreactor and spinner flask constructs, possibly as a response to local shear forces. On the basis of the robustness and reproducibility of the extracellular matrix composition of cartilage constructs, the wavy-walled bioreactor demonstrated promise as an experimental cartilage tissue-engineering vessel. Increased construct growth in the wavy-walled bioreactor may lead to enhanced mechanical properties and expedited in vitro cultivation.
Assuntos
Reatores Biológicos , Cartilagem/fisiologia , Condrócitos/fisiologia , Colágeno Tipo I/biossíntese , Matriz Extracelular/metabolismo , Engenharia Tecidual , Animais , Cartilagem/citologia , Bovinos , Células Cultivadas , Condrócitos/citologia , Estresse Mecânico , Engenharia Tecidual/métodosRESUMO
We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 µm and a resolution of 20 nm, translating to a positional accuracy of less than 3 µm over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.
Assuntos
Reatores Biológicos , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos , Cartilagem Articular , HumanosRESUMO
The design of optimal bioreactor systems for tissue engineering applications requires a sophisticated understanding of the complexities of the bioreactor environment and the role that it plays in the formation of engineered tissues. To this end, a tissue growth model is developed to characterize the tissue growth and extracellular matrix synthesis by chondrocytes seeded and cultivated on polyglycolic acid scaffolds in a wavy-walled bioreactor for a period of 4 weeks. This model consists of four components: (1) a computational fluid dynamics (CFD) model to characterize the complex hydrodynamic environment in the bioreactor, (2) a kinetic growth model to characterize the cell growth and extracellular matrix production dynamics, (3) an artificial neural network (ANN) that empirically correlates hydrodynamic parameters with kinetic constants, and (4) a second ANN that correlates the biochemical composition of constructs with their material properties. In tandem, these components enable the prediction of the dynamics of tissue growth, as well as the final compositional and mechanical properties of engineered cartilage. The growth model methodology developed in this study serves as a tool to predict optimal bioprocessing conditions required to achieve desired tissue properties.
Assuntos
Reatores Biológicos , Cartilagem/crescimento & desenvolvimento , Condrócitos/citologia , Hidrodinâmica , Modelos Biológicos , Engenharia Tecidual/instrumentação , Animais , Simulação por Computador , Desenho de Equipamento , Humanos , Modelos Químicos , Redes Neurais de Computação , Estresse MecânicoRESUMO
Recently, hydrogels (alginate, agarose, polyethylene glycol, etc.) have been investigated as promising cartilage-healing materials. To further improve cell-material interactions or mechanical properties of such hydrogel scaffolds, many materials (such as ceramics or carbon nanotubes) have been added to produce composites with tailored properties. In this study, rosette nanotubes (RNTs, self-assembled nanotubes built from DNA base pairs), hydrogels, and cells (specifically, fibroblast-like type-B synoviocytes [SFB cells] and chondrocytes) were combined via a novel electrospinning technique to generate three-dimensional implantable scaffolds for cartilage repair. Importantly, results of this study showed that electrospun RNT/hydrogel composites improved both SFB cell and chondrocyte functions. RNT/hydrogel composites promoted SFB cell chondrogenic differentiation in 2 week culture experiments. Further, studies demonstrated that RNTs enhanced hydrogel adhesive strength to severed collagen. Results of this study thus provided a nanostructured scaffold that enhanced SFB cell adhesion, viability, and chondrogenic differentiation compared to nanosmooth hydrogels without RNTs. This study provided an alternative cartilage regenerative material derived from RNTs that could be directly electrospun into cartilage defects (with SFB cells and/or chondrocytes) to bond to severed collagen and promote cell adhesion, viability, and subsequent functions.
Assuntos
Cartilagem/citologia , Hidrogéis/síntese química , Nanotubos/química , Engenharia Tecidual/instrumentação , Alicerces Teciduais/química , Adesividade , Animais , Cartilagem/fisiologia , Técnicas de Cultura de Células , Resinas Compostas/síntese química , Resinas Compostas/química , Galvanoplastia/métodos , Feminino , Fibroblastos/citologia , Fibroblastos/fisiologia , Hidrogéis/química , Teste de Materiais , Modelos Biológicos , Modelos Moleculares , Suínos , Membrana Sinovial/citologia , Membrana Sinovial/fisiologiaRESUMO
Functional engineered cartilage constructs represent a promising therapeutic approach for the replacement of damaged articular cartilage. The in vitro generation of cartilage tissue suitable for repair requires an understanding of the complex interrelationships between environmental cues, such as hydrodynamic forces, and tissue growth and development. In the present study, engineered cartilage constructs were cultivated in four well-defined hydrodynamic environments within a bioreactor, and correlations were established between construct ultrastructural and mechanical properties and key hydrodynamic parameters. Results suggest that even for similar composition, constructs may exhibit different mechanical properties due to differences in their ultrastructure that can be modulated by hydrodynamic parameters. For example, improved mechanical properties were observed in constructs that exhibited a thick fibrous outer capsule as a result of cultivation under increased hydrodynamic shear. In particular, uniformity in the contribution of the fluid velocity vectors (axial, radial, and tangential) to the total fluid velocity and shear stress were the hydrodynamic parameters that most affected the construct properties under investigation. The correlations identified here may be useful in the development of engineered tissue growth models that inform the design of bioreactor cultivation systems toward the production of clinically relevant engineered cartilage.
Assuntos
Cartilagem , Engenharia Tecidual/métodos , Animais , Reatores Biológicos , Bovinos , Microscopia , Modelos Teóricos , Estresse Mecânico , Engenharia Tecidual/instrumentaçãoRESUMO
Bioreactors have played a crucial role in recent approaches to cartilage tissue engineering, providing an environment that promotes efficient cell seeding, nutrient and waste transport, and essential physical stimuli. This study employed a wavy-walled bioreactor to investigate the effects of the hydrodynamic environment on the properties of engineered cartilage. Its unique design provides multiple hydrodynamic environments within one setting. A tissue growth model was developed to characterize the tissue growth and extracellular matrix synthesis by chondrocytes seeded and cultivated on polyglycolic acid scaffolds in the wavy-walled bioreactor for a period of 4 weeks. This model consists of four components: 1) a computational fluid dynamics model, 2) a kinetic growth model, 3) an artificial neural network that empirically correlates hydrodynamic parameters with kinetic constants, and 4) a second artificial neural network that correlates the biochemical composition of constructs with their material properties. In tandem, these components enable the prediction of the dynamics of tissue growth, as well as the final compositional and mechanical properties of engineered cartilage. The growth model methodology developed in this study serves as a tool to predict the optimal bioprocessing conditions required to achieve desired tissue properties.
Assuntos
Reatores Biológicos , Engenharia Tecidual/métodos , Animais , Bovinos , Células Cultivadas , Condrócitos/citologia , Cinética , Modelos Teóricos , Redes Neurais de Computação , Reprodutibilidade dos TestesRESUMO
Synovial membrane has been shown to contain mesenchymal stem cells. We hypothesized that an enriched population of synovial fibroblasts would undergo chondrogenic differentiation and secrete cartilage extracellular matrix to a greater extent than would a mixed synovial cell population (MSCP). The optimum doses of transforming growth factor beta 1 (TGF-beta1) and insulin-like growth factor 1 (IGF-1) for chondrogenesis were investigated. CD14-negative isolation was used to obtain a porcine cell population enriched in type-B synovial fibroblasts (SFB) from an MSCP. The positive cell surface markers in SFB were CD90, CD44, and cadherin-11. SFB and MSCP were cultured in the presence of 20 ng/mL TGF-beta1 for 7 days, and SFB were demonstrated to have higher chondrogenic potential. Further dose-response studies were carried out using the SFB cells and several doses of TGF-beta1 (2, 10, 20, and 40 ng/mL) and/or IGF-1 (1, 10, 100, and 500 ng/mL) for 14 days. TGF-beta1 supplementation was essential for chondrogenesis and prevention of cell death, whereas IGF-1 did not have a significant effect on the SFB cell number or glycosaminoglycan production. This study demonstrates that the CD14-negative isolation yields an enhanced cell population SFB that is more potent than MSCP as a cell source for cartilage tissue engineering.
Assuntos
Condrogênese/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Receptores de Hialuronatos/metabolismo , Membrana Sinovial/citologia , Membrana Sinovial/fisiopatologia , Fator de Crescimento Transformador beta1/farmacologia , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Condrogênese/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Suínos , Membrana Sinovial/efeitos dos fármacos , Engenharia Tecidual/métodos , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Physical forces experienced by engineered-tissues during in vitro cultivation influence tissue growth and function. The hydrodynamic environment within bioreactors plays a decisive role in providing the necessary physical stimuli and nutrient transport to support tissue development. Our overall goal is to investigate interrelationships between the local hydrodynamic environment in the bioreactor and the structural and functional tissue properties in order to optimize the production of clinically relevant engineered-tissues. To this end, we used computational fluid dynamics (CFD) modeling to characterize the complex hydrodynamic environment in a wavy-walled bioreactor used for cultivation of tissue-engineered cartilage constructs and examined the changes in the flow field due to the presence of constructs. The flow-induced shear stress range experienced by engineered constructs cultivated in the wavy-walled bioreactor (0-0.67 dyn/cm(2)) was found to be significantly lower than that in the spinner flask (0-1.2 dyn/cm(2)), and to be modulated by the radial or axial position of the constructs. These CFD results are validated by experimental particle-image velocimetry (PIV) measurements previously reported by our group. Results from the present study indicate that the location of constructs in the bioreactor not only affected the magnitude and distribution of the shear stresses on the constructs, but also other hydrodynamic parameters, such as the directional distribution of the fluid velocity and the degree of fluid recirculation, all of which may differentially influence the development of tissue-engineered constructs.
Assuntos
Reatores Biológicos , Técnicas de Cultura de Células/instrumentação , Regeneração Tecidual Guiada/instrumentação , Microfluídica/instrumentação , Modelos Teóricos , Engenharia Tecidual/instrumentação , Técnicas de Cultura de Células/métodos , Desenho Assistido por Computador , Ecossistema , Desenho de Equipamento , Análise de Falha de Equipamento , Regeneração Tecidual Guiada/métodos , Microfluídica/métodos , Pressão , Engenharia Tecidual/métodosRESUMO
In vitro cartilage tissue engineering culture systems benefit from a fine balance of biochemical and mechanical components to maintain the chondrocyte phenotype. This balance, however, can be disrupted by using typical methods for cultivating chondrogenic cells in medium supplemented with fetal bovine serum (FBS) and growth factors. Our goal was to determine the effects of fluid-dynamic stimuli, fetal bovine serum and dexamethasone on the chondrogenesis of 14-day synoviocyte pellet cultures in the presence of TGF-beta1. We employed a pellet culture system that provides a highly cellular three-dimensional structure that permits differentiation and extracellular matrix synthesis. Our results indicated that FBS inhibited glycosaminoglycan (GAG) and type II collagen production. Interestingly, the effect of dynamic stimuli was modulated by the presence of FBS; mixed serum-free cultures had increased GAG production, whereas mixed cultures with 10% FBS exhibited less GAG production compared with their static counterparts, possibly due to pronounced suppressive effects of FBS via increased transport. Dexamethasone addition during the first week of culture resulted in enhanced extracellular matrix production and increased cellularity. Moreover, the presence of 10% FBS in addition to ITS(+) and TGF-beta1 did not significantly increase cell proliferation compared with serum-free medium. These results indicate the importance of a comprehensive analysis of growth conditions for each cell culture system.
Assuntos
Condrogênese/efeitos dos fármacos , Dexametasona/farmacologia , Soro , Membrana Sinovial/citologia , Membrana Sinovial/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Animais , Bovinos , Separação Celular , Células Cultivadas , Meios de Cultura , Feminino , SuínosRESUMO
Cartilage tissue engineering requires the use of bioreactors in order to enhance nutrient transport and to provide sufficient mechanical stimuli to promote extracellular matrix (ECM) synthesis by chondrocytes. The amount and quality of ECM components is a large determinant of the biochemical and mechanical properties of engineered cartilage constructs. Mechanical forces created by the hydrodynamic environment within the bioreactors are known to influence ECM synthesis. The present study characterizes the hydrodynamic environment within a novel wavy-walled bioreactor (WWB) used for the development of tissue-engineered cartilage. The geometry of this bioreactor provides a unique hydrodynamic environment for mammalian cell and tissue culture, and investigation of hydrodynamic effects on tissue growth and function. The flow field within the WWB was characterized using two-dimensional particle-image velocimetry (PIV). The flow in the WWB differed significantly from that in the traditional spinner flask both qualitatively and quantitatively, and was influenced by the positioning of constructs within the bioreactor. Measurements of velocity fields were used to estimate the mean-shear stress, Reynolds stress, and turbulent kinetic energy components in the vicinity of the constructs within the WWB. The mean-shear stress experienced by the tissue-engineered constructs in the WWB calculated using PIV measurements was in the range of 0-0.6 dynes/cm2. Quantification of the shear stress experienced by cartilage constructs, in this case through PIV, is essential for the development of tissue-growth models relating hydrodynamic parameters to tissue properties.
Assuntos
Reatores Biológicos , Biotecnologia/instrumentação , Engenharia Tecidual/métodos , Animais , Biotecnologia/métodos , Cartilagem/metabolismo , Células Cultivadas , Condrócitos/citologia , Desenho de Equipamento , Matriz Extracelular , Humanos , Mecanotransdução Celular , Estresse Mecânico , Engenharia Tecidual/instrumentaçãoRESUMO
The wavy-walled bioreactor (WWB) possesses a novel geometry comprised of walls with sinusoidal waves that mimic baffles in an effort to promote mixing. This geometry provides a unique hydrodynamic environment suitable for the cultivation of mammalian cells and tissues and the investigation of fluid mechanical effects on cell and tissue growth and development. In the present study, mixing in WWB was characterized and compared to that in a conventional spinner flask (SF). The key parameters included in this characterization were mixing time, residence time distribution (RTD), and dissolved oxygen concentration during engineered cartilage tissue cultivation. Factors that influenced mixing in WWB included wave amplitude, agitation rate, and the ratio of the impeller diameter to the tank diameter (D/T). Data obtained from RTD and acid base neutralization studies confirmed the presence of different mixing zones in WWB. A theoretical comparison of WWB to a baffled spinner flask (BSF) using computational fluid dynamics (CFD) modeling predicted that while enhanced mixing was achieved in wavy-walled and BSF bioreactors, the shear stresses applied on tissue constructs were 15% lower in WWB. Improved mixing was achieved in WWB compared to the SF at similar D/T ratios, verified by improved oxygen transport and increased dispersion. However, for lower D/T ratios mixing in WWB was not necessarily improved. This study demonstrated the importance of characterization of mixing by showing the impact of even minor changes in bioreactor geometry and operating conditions.
Assuntos
Reatores Biológicos , Modelos Teóricos , Engenharia Tecidual/instrumentaçãoRESUMO
A novel wavy-walled bioreactor designed to enhance mixing at controlled shear stress levels was used to culture chondrocytes in suspension. Chondrocyte aggregation in suspensions mixed at 30, 50, and 80 rpm was characterized in the wavy-walled bioreactor and compared with that in conventional smooth-walled and baffled-walled spinner flask bioreactors. Aggregation was characterized in terms of the percentage of cells that aggregated over time, and aggregate size changes over time. The kinetics of chondrocyte aggregation observed in the bioreactors was composed of two phases: early aggregation between 0 and 2 h of culture, and late aggregation between 3 and 24 h of culture. At 50 rpm, the kinetics of early aggregation in the wavy-walled bioreactor was approximately 25% and 65% faster, respectively, than those in the smooth-walled and baffled-walled spinner flask bioreactors. During the late aggregation phase, the kinetics of aggregation in the wavy-walled bioreactor were approximately 45% and 65% faster, respectively, than in the smooth-walled and baffled-walled spinner flasks. The observed improved kinetics of chondrocyte aggregation was obtained at no cost to the cell survival rate. Results of computerized image analysis suggest that chondrocyte aggregation occurred initially by the formation of new aggregates via cell-cell interactions and later by the joining of small aggregates into larger cell clumps. Aggregates appeared to grow for only a couple of hours in culture before reaching a steady size, possibly determined by limitations imposed by the hydrodynamic environment. These results suggest that the novel geometry of the wavy-walled bioreactor generates a hydrodynamic environment distinct from those traditionally used to culture engineered cartilage. Such differences may be useful in studies aimed at distinguishing the effects of the hydrodynamic environment on tissue-engineered cartilage. Characterizing the wavy-walled bioreactor's hydrodynamic environment and its effects on cartilage cell/tissue culture can help establish direct relationships between hydrodynamic forces and engineered tissue properties.