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Articular cartilage defects have limited healing potential and, when left untreated, can lead to osteoarthritis. Tissue engineering focuses on regenerating the damaged joint surface, preferably in an early stage. Here, we investigate the regenerative potential of three-dimensional (3D) constructs consisting of human induced pluripotent stem cell (iPSC)-derived chondrocytes in gelatin methacryloyl (GelMA) hydrogel for stable hyaline cartilage production. iPSC-derived chondrocytes are encapsulated in GelMA hydrogel at low (1 × 107 ml-1 ) and high (2 × 107 ml-1 ) density. In a conventional medium, GelMA hydrogel supports the chondrocyte phenotype, as opposed to cells cultured in 3D in absence of hydrogel. Moreover, encapsulated iPSC-derived chondrocytes preserve their in vivo matrix formation capacity after 21 days in vitro. In differentiation medium, hyaline cartilage-like tissue forms after 21 days, demonstrated by highly sulfated glycosaminoglycans and collagen type II. Matrix deposition is delayed at low encapsulation density, corroborating with lower transcript levels of COL2A1. An ectopic assay in nude mice demonstrates further maturation of the matrix deposited in vitro. Direct ectopic implantation of iPSC-derived chondrocyte-laden GelMA, without in vitro priming, also generates hyaline cartilage-like tissue, albeit less mature. Since it is unclear what maturity upon implantation is desired for joint surface regeneration, this is an attractive technology to generate immature and more mature hyaline cartilage-like tissue.
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Cartílago Articular , Células Madre Pluripotentes Inducidas , Animales , Condrocitos , Gelatina , Humanos , Hidrogeles , Metacrilatos , Ratones , Ratones Desnudos , Ingeniería de Tejidos/métodosRESUMEN
Osteochondral defects are deep joint surface lesions that affect the articular cartilage and the underlying subchondral bone. In the current study, a tissue engineering approach encompassing individual cells encapsulated in a biocompatible hydrogel is explored in vitro and in vivo. Cell-laden hydrogels containing either human periosteum-derived progenitor cells (PDCs) or human induced pluripotent stem cell (iPSC)-derived chondrocytes encapsulated in gelatin methacryloyl (GelMA) were evaluated for their potential to regenerate the subchondral mineralized bone and the articular cartilage on the joint surface, respectively. PDCs are easily isolated and expanded progenitor cells that are capable of generating mineralized cartilage and bone tissue in vivo via endochondral ossification. iPSC-derived chondrocytes are an unlimited source of stable and highly metabolically active chondrocytes. Cell-laden hydrogel constructs were cultured for up to 28 days in a serum-free chemically defined chondrogenic medium. On day 1 and day 21 of the differentiation period, the cell-laden constructs were implanted subcutaneously in nude mice to evaluate ectopic tissue formation 4 weeks post-implantation. Taken together, the data suggest that iPSC-derived chondrocytes encapsulated in GelMA can generate hyaline cartilage-like tissue constructs with different levels of maturity, while using periosteum-derived cells in the same construct type generates mineralized tissue and cortical bone in vivo. Therefore, the aforementioned cell-laden hydrogels can be an important part of a multi-component strategy for the manufacturing of an osteochondral implant.
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While available treatments have addressed a variety of complications in the dentoalveolar region, associated challenges have resulted in exploration of tissue engineering techniques. Often, scaffold biomaterials with specific properties are required for such strategies to be successful, development of which is an active area of research. This study focuses on the development of a copolymer of poly (N-isopropylacrylamide) (pNIPAM) and chitosan, used for 3D printing of scaffolds for dentoalveolar regeneration. The synthesized material was characterized by Fourier transform infrared spectroscopy, and the possibility of printing was evaluated through various printability tests. The rate of degradation and swelling was analyzed through gravimetry, and surface morphology was characterized by scanning electron microscopy. Viability of dental pulp stem cells seeded on the scaffolds was evaluated by live/dead analysis and DNA quantification. The results demonstrated successful copolymerization, and three formulations among various synthesized formulations were successfully 3D printed. Up to 35% degradability was confirmed within 7 days, and a maximum swelling of approximately 1200% was achieved. Furthermore, initial assessment of cell viability demonstrated biocompatibility of the developed scaffolds. While further studies are required to achieve the tissue engineering goals, the present results tend to indicate that the proposed hydrogel might be a valid candidate for scaffold fabrication serving dentoalveolar tissue engineering through 3D printing.
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Incorporation of growth factors, signaling molecules and drugs can be vital for the success of tissue engineering in complex structures such as the dentoalveolar region. This has led to the development of a variety of drug release systems. This study aimed to develop pNIPAM-methylcellulose microgels with different synthesis parameters based on a 23 full factorial design of experiments for this application. Microgel properties, including volume phase transition temperature (VPTT), hydrodynamic size, drug loading and release, and cytocompatibility were systematically evaluated. The results demonstrated successful copolymerization and development of the microgels, a hydrodynamic size ranging from â¼200 to â¼500 nm, and VPTT in the range of 34-39 °C. Furthermore, loading of genipin, capable of inducing odontoblastic differentiation, and its sustained release over a week was shown in all formulations. Together, this can serve as a solid basis for the development of tunable drug-delivering pNIPAM-methylcellulose microgels for specific tissue engineering applications.
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Microgeles , Geles/química , Metilcelulosa , Ingeniería de Tejidos , Temperatura de TransiciónRESUMEN
Osteoclasts are specialized multinucleated cells with the unique capacity to resorb bone. Despite insight into the various steps of the interaction of osteoclast precursors leading to osteoclast formation, surprisingly little is known about what happens with the multinucleated cell itself after it has been formed. Is fusion limited to the short period of its formation, or do osteoclasts have the capacity to change their size and number of nuclei at a later stage? To visualize these processes we analyzed osteoclasts generated in vitro with M-CSF and RANKL from mouse bone marrow and native osteoclasts isolated from rabbit bones by live cell microscopy. We show that osteoclasts fuse not only with mononuclear cells but also with other multinucleated cells. The most intriguing finding was fission of the osteoclasts. Osteoclasts were shown to have the capacity to generate functional multinucleated compartments as well as compartments that contained apoptotic nuclei. These compartments were separated from each other, each giving rise to a novel functional osteoclast or to a compartment that contained apoptotic nuclei. Our findings suggest that osteoclasts have the capacity to regulate their own population in number and function, probably to adapt quickly to changing situations.
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Osteoclastos/metabolismo , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/ultraestructura , Resorción Ósea/patología , Diferenciación Celular , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Osteoclastos/citología , Ligando RANK , ConejosRESUMEN
Spheroids are widely applied as building blocks for biofabrication of living tissues, where they exhibit spontaneous fusion toward an integrated structure upon contact. Tissue fusion is a fundamental biological process, but due to a lack of automated monitoring systems, the in-depth characterization of this process is still limited. Therefore, a quantitative high-throughput platform was developed to semi-automatically select doublet candidates and automatically monitor their fusion kinetics. Spheroids with varying degrees of chondrogenic maturation (days 1, 7, 14, and 21) were produced from two different cell pools, and their fusion kinetics were analyzed via the following steps: (1) by applying a novel spheroid seeding approach, the background noise was decreased due to the removal of cell debris while a sufficient number of doublets were still generated. (2) The doublet candidates were semi-automatically selected, thereby reducing the time and effort spent on manual selection. This was achieved by automatic detection of the microwells and building a random forest classifier, obtaining average accuracies, sensitivities, and precisions ranging from 95.0% to 97.4%, from 51.5% to 92.0%, and from 66.7% to 83.9%, respectively. (3) A software tool was developed to automatically extract morphological features such as the doublet area, roundness, contact length, and intersphere angle. For all data sets, the segmentation procedure obtained average sensitivities and precisions ranging from 96.8% to 98.1% and from 97.7% to 98.8%, respectively. Moreover, the average relative errors for the doublet area and contact length ranged from 1.23% to 2.26% and from 2.30% to 4.66%, respectively, while the average absolute errors for the doublet roundness and intersphere angle ranged from 0.0083 to 0.0135 and from 10.70 to 13.44°, respectively. (4) The data of both cell pools were analyzed, and an exponential model was used to extract kinetic parameters from the time-series data of the doublet roundness. For both cell pools, the technology was able to characterize the fusion rate and quality in an automated manner and allowed us to demonstrate that an increased chondrogenic maturity was linked with a decreased fusion rate. The platform is also applicable to other spheroid types, enabling an increased understanding of tissue fusion. Finally, our approach to study spheroid fusion over time will aid in the design of controlled fabrication of "assembloids" and bottom-up biofabrication of living tissues using spheroids.
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Dentoalveolar tissue engineering is an emerging yet challenging field, considering the lack of suitable materials and difficulty to produce patient-specific hydrogel scaffolds. The present paper aims to produce a 3D printable and tuneable biomaterial by copolymerizing a synthesized water-soluble chitosan derivative called maleic anhydride grafted chitosan (MA-C) with gelatin using genipin, a natural crosslinking agent. Development and testing of this material for 3D printing, degradation, and swelling demonstrated the ability to fabricate scaffolds with controlled physical properties based on pre-determined designs. The MA-C-gelatin copolymer demonstrated excellent biocompatibility, which was verified by analyzing the viability, growth and proliferation of human dental pulp stem cells seeded on MA-C-gelatin constructs through live/dead, alamar blue and DNA quantification assays. Based on the present findings, the proposed material might be a suitable candidate for dentoalveolar tissue engineering, while further research is required to achieve this goal.
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Quitosano , Proliferación Celular , Gelatina , Humanos , Polímeros , Regeneración , Andamios del TejidoRESUMEN
Methacryloyl gelatin (GelMA) is a versatile material for bioprinting because of its tunable physical properties and inherent bioactivity. Bioprinting of GelMA is often met with challenges such as lower viscosity of GelMA inks due to higher methacryloyl substitution and longer physical gelation time at room temperature. In this study, a tunable interpenetrating polymer network (IPN) hydrogel was prepared from gelatin-hyaluronan dialdehyde (Gel-HDA) Schiff's polymer, and 100% methacrylamide substituted GelMA for biofabrication through extrusion based bioprinting. Temperature sweep rheology measurements show a higher sol-gel transition temperature for IPN (30 °C) compared to gold standard GelMA (27 °C). Furthermore, to determine the tunability of the IPN hydrogel, several IPN samples were prepared by combining different ratios of Gel-HDA and GelMA achieving a compressive modulus ranging from 20.6 ± 2.48 KPa to 116.7 ± 14.80 KPa. Our results showed that the mechanical properties and printability at room temperature could be tuned by adjusting the ratios of GelMA and Gel-HDA. To evaluate cell response to the material, MC3T3-E1 mouse pre-osteoblast cells were embedded in hydrogels and 3D-printed, demonstrating excellent cell viability and proliferation after 10 d of 3Din vitroculture, making the IPN an interesting bioink for the fabrication of 3D constructs for tissue engineering applications.
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Bioimpresión , Gelatina , Animales , Ácido Hialurónico , Hidrogeles , Metacrilatos , Ratones , Polímeros , Impresión Tridimensional , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
The balance between bone formation and bone resorption in inflammatory diseases is often disturbed. Periodontitis, a chronic inflammation of the tooth gums, leads to unwanted bone loss as a response to inflammatory compounds such as interleukin-1ß (IL-1ß). This excessive bone loss reflects an increased osteoclast formation and activity. Osteoclast formation is a multistep process driven by osteoclastogenesis supporting cells such as periodontal ligament fibroblasts. The inflammatory factors can induce osteoclastogenesis, probably also by affecting the periodontal ligament fibroblast. In this study we investigated how pre-culture of periodontal ligament fibroblasts with IL-1ß affected osteoclastogenesis. Fibroblasts were pre-cultured with IL-1ß and/or dexamethasone, a commonly used anti-inflammatory compound, before being co-cultured with peripheral blood mononuclear cells (PBMCs). Pre-culture with IL-1ß (1-100 ng/ml) resulted in an increased number of adhered PBMCs as well as an increased mRNA expression of intercellular adhesion molecule-1 (ICAM-1), macrophage colony stimulating factor (M-CSF) and IL-1ß. Pre-culture with IL-1ß also caused retraction of fibroblasts and an augmented formation of TRACP(+) multinucleated cells. Our data suggest that stimulation of fibroblasts with IL-1ß has a long-lasting effect, leading to a significantly increased osteoclastogenesis. These results provide new insights for understanding excessive bone loss in periodontitis.
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Fibroblastos/citología , Interleucina-1beta/fisiología , Osteoclastos/citología , Ligamento Periodontal/citología , Adhesión Celular/efectos de los fármacos , Diferenciación Celular , Células Cultivadas , Técnicas de Cocultivo , Dexametasona/farmacología , Fibroblastos/efectos de los fármacos , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-1beta/farmacología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/fisiología , Factor Estimulante de Colonias de Macrófagos/metabolismo , Osteoclastos/efectos de los fármacos , Transcripción GenéticaRESUMEN
Nitric oxide (NOâ¢) is a free radical gas, produced in the human body to regulate physiological processes, such as inflammatory and immune responses. It is required for skin health; therefore, a lack of NO⢠is known to cause or worsen skin conditions related to three biomedical applications- infection treatment, injury healing, and blood circulation. Therefore, research on its topical release has been increasing for the last two decades. The storage and delivery of nitric oxide in physiological conditions to compensate for its deficiency is achieved through pharmacological compounds called NO-donors. These are further incorporated into scaffolds to enhance therapeutic treatment. A wide range of polymeric scaffolds has been developed and tested for this purpose. Hence, this review aims to give a detailed overview of the natural, synthetic, and semisynthetic polymeric matrices that have been evaluated for antimicrobial, wound healing, and circulatory dermal applications. These matrices have already set a solid foundation in nitric oxide release and their future perspective is headed toward an enhanced controlled release by novel functionalized semisynthetic polymer carriers and co-delivery synergetic platforms. Finally, further clinical tests on patients with the targeted condition will hopefully enable the eventual commercialization of these systems.
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Although current treatments can successfully address a wide range of complications in the dentoalveolar region, they often still suffer from drawbacks and limitations, resulting in sub-optimal treatments for specific problems. In recent decades, significant progress has been made in the field of tissue engineering, aiming at restoring damaged tissues via a regenerative approach. Yet, the translation into a clinical product is still challenging. Novel technologies such as bioprinting have been developed to solve some of the shortcomings faced in traditional tissue engineering approaches. Using automated bioprinting techniques allows for precise placement of cells and biological molecules and for geometrical patient-specific design of produced biological scaffolds. Recently, bioprinting has also been introduced into the field of dentoalveolar tissue engineering. However, the choice of a suitable material to encapsulate cells in the development of so-called bioinks for bioprinting dentoalveolar tissues is still a challenge, considering the heterogeneity of these tissues and the range of properties they possess. This review, therefore, aims to provide an overview of the current state of the art by discussing the progress of the research on materials used for dentoalveolar bioprinting, highlighting the advantages and shortcomings of current approaches and considering opportunities for further research.
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Research in bioprinting is booming due to its potential in addressing several manufacturing challenges in regenerative medicine. However, there are still many hurdles to overcome to guarantee cell survival and good printability. For the 3D extrusion-based bioprinting, cell viability is amongst one of the lowest of all the bioprinting techniques and is strongly influenced by various factors including the shear stress in the print nozzle. The goal of this study is to quantify, by means of in silico modeling, the mechanical environment experienced by the bioink during the printing process. Two ubiquitous nozzle shapes, conical and blunted, were considered, as well as three common hydrogels with material properties spanning from almost Newtonian to highly shear-thinning materials following the power-law behavior: Alginate-Gelatin, Alginate and PF127. Comprehensive in silico testing of all combinations of nozzle geometry variations and hydrogels was achieved by combining a design of experiments approach (DoE) with a computational fluid dynamics (CFD) of the printing process, analyzed through a machine learning approach named Gaussian Process. Available experimental results were used to validate the CFD model and justify the use of shear stress as a surrogate for cell survival in this study. The lower and middle nozzle radius, lower nozzle length and the material properties, alone and combined, were identified as the major influencing factors affecting shear stress, and therefore cell viability, during printing. These results were successfully compared with those of reported experiments testing viability for different nozzle geometry parameters under constant flow rate or constant pressure. The in silico 3D bioprinting platform developed in this study offers the potential to assist and accelerate further development of 3D bioprinting.
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The formation of bone resorbing osteoclasts in vivo is orchestrated by cells of the osteoblast lineage such as periodontal ligament fibroblasts that provide the proper signals to osteoclast precursors. Although the requirement of cell-cell interactions is widely acknowledged, it is unknown whether these interactions influence the expression of genes required for osteoclastogenesis and the ultimate formation of osteoclasts. In the present study we investigated the effect of cell-cell interaction on the mRNA expression of adhesion molecules and molecules involved in osteoclast formation in cultures of peripheral blood mononuclear cells (PBMCs) and human primary periodontal ligament fibroblasts, both as solitary cultures and in co-culture. We further analyzed the formation of multinucleated, tartrate resistant acid phosphatase (TRACP) positive cells and assessed their bone resorbing abilities. Interestingly, gene expression of intercellular adhesion molecule-1 (ICAM-1) and of osteoclastogenesis-related genes (RANKL, RANK, TNF-alpha, and IL-1beta) was highly up-regulated in the co-cultures compared to mono-cultures and the 5-10-fold up-regulation reflected a synergistic increase due to direct cell-cell interaction. This induction strongly overpowered the effects of known osteoclastogenesis inducers 1,25(OH)(2)VitD(3) and dexamethasone. In case of indirect cell-cell contact mRNA expression was not altered, indicating that heterotypic adhesion is required for the increase in gene expression. In addition, the number of osteoclast-like cells that were formed in co-culture with periodontal ligament fibroblasts was significantly augmented compared to mono-cultures. Our data indicate that cell-cell adhesion between osteoclast precursors and periodontal ligament fibroblasts significantly modulates the cellular response which favors the expression of osteoclast differentiation genes and the ultimate formation of osteoclasts.
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Adhesión Celular/genética , Comunicación Celular/genética , Diferenciación Celular/genética , Fibroblastos/metabolismo , Osteoclastos/metabolismo , Ligamento Periodontal/metabolismo , Fosfatasa Ácida/metabolismo , Resorción Ósea/genética , Calcitriol/farmacología , Adhesión Celular/efectos de los fármacos , Comunicación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Dexametasona/farmacología , Fibroblastos/efectos de los fármacos , Regulación de la Expresión Génica , Humanos , Molécula 1 de Adhesión Intercelular/genética , Interleucina-1beta/genética , Isoenzimas/metabolismo , Osteoclastos/efectos de los fármacos , Ligamento Periodontal/citología , Ligamento Periodontal/efectos de los fármacos , Ligando RANK/genética , ARN Mensajero/metabolismo , Receptor Activador del Factor Nuclear kappa-B/genética , Fosfatasa Ácida Tartratorresistente , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Adhesion between osteoblasts and osteoclast precursors is established via an interaction involving intercellular adhesion molecule-1 (ICAM-1) on osteoblasts and leukocyte function-associated antigen-1 (LFA-1) on osteoclast precursors. The latter cells also express ICAM-1, but little is known about the expression over time and its possible role during osteoclastogenesis. In the present study we investigated the expression of ICAM-1 on both human osteoblast-like cells and osteoclast precursors in a co-culture. The protein expression on osteoclast precursors strongly increased whereas the osteoblast-like cells became ICAM-1 negative. Interestingly, ICAM-1 on osteoclast precursors manifested as clusters which localized at the baso-lateral membrane. Furthermore, clustered ICAM-1 was associated with F-actin and remained present for several days. Our data suggest that osteoblastic ICAM-1 is mainly involved in the initial adhesion of osteoclast precursors whereas clustered ICAM-1 on osteoclast precursors and its association with F-actin suggest an involvement in cell movement at a later stage.
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Diferenciación Celular , Molécula 1 de Adhesión Intercelular/metabolismo , Osteoclastos/citología , Células Madre/citología , Actinas/metabolismo , Adhesión Celular , Movimiento Celular , Células Cultivadas , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/genética , Osteoclastos/metabolismo , Osteoclastos/fisiología , Células Madre/metabolismo , Células Madre/fisiologíaRESUMEN
Studies on monolayer cultures and whole-animal models for the prediction of the response of native human tissue are associated with limitations. Therefore, more and more laboratories are tending towards multicellular spheroids grown in vitro as a model of native tissues. In addition, they are increasingly used in a wide range of biofabrication methodologies. These 3D microspheroids are generated through a self-assembly process that is still poorly characterised, called cellular aggregation. Here, a system is proposed for the automated, non-invasive and high throughput monitoring of the morphological changes during cell aggregation. Microwell patterned inserts were used for spheroid formation while an automated microscope with 4x bright-field objective captured the morphological changes during this process. Subsequently, the acquired time-lapse images were automatically segmented and several morphological features such as minor axis length, major axis length, roundness, area, perimeter and circularity were extracted for each spheroid. The method was quantitatively validated with respect to manual segmentation on four sets of ± 60 spheroids. The average sensitivities and precisions of the proposed segmentation method ranged from 96.67-97.84% and 96.77-97.73%, respectively. In addition, the different morphological features were validated, obtaining average relative errors between 0.78-4.50%. On average, a spheroid was processed 73 times faster than a human operator. As opposed to existing algorithms, our methodology was not only able to automatically monitor compact spheroids but also the aggregation process of individual spheroids, and this in an accurate and high-throughput manner. In total, the aggregation behaviour of more than 700 individual spheroids was monitored over a duration of 16 hours with a time interval of 5 minutes, and this could be increased up to 48,000 for the described culture format. In conclusion, the proposed system has the potential to be used for unravelling the mechanisms involved in spheroid formation and monitoring their formation during large-scale manufacturing protocols.
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Procesamiento de Imagen Asistido por Computador/métodos , Periostio/citología , Células Madre/citología , Células Cultivadas , Humanos , Periostio/metabolismo , Esferoides Celulares/metabolismo , Células Madre/metabolismoRESUMEN
Clinical translation of cell-based strategies for regenerative medicine demands predictable in vivo performance where the use of sera during in vitro preparation inherently limits the efficacy and reproducibility. Here, we present a bioinspired approach by serum-free pre-conditioning of human periosteum-derived cells, followed by their assembly into microaggregates simultaneously primed with bone morphogenetic protein 2 (BMP-2). Pre-conditioning resulted in a more potent progenitor cell population, while aggregation induced osteochondrogenic differentiation, further enhanced by BMP-2 stimulation. Ectopic implantation displayed a cascade of events that closely resembled the natural endochondral process resulting in bone ossicle formation. Assessment in a critical size long-bone defect in immunodeficient mice demonstrated successful bridging of the defect within 4 weeks, with active contribution of the implanted cells. In short, the presented serum-free process represents a biomimetic strategy, resulting in a cartilage tissue intermediate that, upon implantation, robustly leads to the healing of a large long-bone defect.
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Fracturas Óseas/metabolismo , Fracturas Óseas/patología , Periostio/citología , Trasplante de Células Madre , Células Madre/citología , Células Madre/metabolismo , Cicatrización de Heridas , Animales , Antígenos CD34/metabolismo , Biomarcadores , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Regeneración Ósea , Diferenciación Celular , Condrogénesis/genética , Modelos Animales de Enfermedad , Fracturas Óseas/terapia , Humanos , Ratones , Ratones Noqueados , Osteogénesis/genética , Agregado de Proteínas , Transducción de SeñalRESUMEN
Bone tissue engineering (TE) aims to develop reproducible and predictive three-dimensional (3D) TE constructs, defined as cell-seeded scaffolds produced by a controlled in vitro process, to heal or replace damaged and nonfunctional bone. To control and assure the quality of the bone TE constructs, a prerequisite for regulatory authorization, there is a need to develop noninvasive analysis techniques to evaluate TE constructs and to monitor their behavior in real time during in vitro culturing. Most analysis techniques, however, are limited to destructive end-point analyses. This study investigates the use of the nontoxic alamarBlue(®) (AB) reagent, which is an indicator for metabolic cell activity, for monitoring the cellularity of 3D TE constructs in vitro as part of a bioreactor culturing processes. Within the field of TE, bioreactors have a huge potential in the translation of TE concepts to the clinic. Hence, the use of the AB reagent was evaluated not only in static cultures, but also in dynamic cultures in a perfusion bioreactor setup. Hereto, the AB assay was successfully integrated in the bioreactor-driven TE construct culture process in a noninvasive way. The obtained results indicate a linear correlation between the overall metabolic activity and the total DNA content of a scaffold upon seeding as well as during the initial stages of cell proliferation. This makes the AB reagent a powerful tool to follow-up bone TE constructs in real-time during static as well as dynamic 3D cultures. Hence, the AB reagent can be successfully used to monitor and predict cell confluence in a growing 3D TE construct.
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Huesos/fisiología , Sistemas de Computación , Oxazinas/metabolismo , Técnicas de Cultivo de Tejidos/métodos , Ingeniería de Tejidos/métodos , Xantenos/metabolismo , Reactores Biológicos , Huesos/efectos de los fármacos , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Perfusión , Factores de Tiempo , Andamios del Tejido , Titanio/farmacologíaRESUMEN
Cell seeding into scaffolds plays a crucial role in the development of efficient bone tissue engineering constructs. Hence, it becomes imperative to identify the key factors that quantitatively predict reproducible and efficient seeding protocols. In this study, the optimization of a cell seeding process was investigated using design of experiments (DOE) statistical methods. Five seeding factors (cell type, scaffold type, seeding volume, seeding density, and seeding time) were selected and investigated by means of two response parameters, critically related to the cell seeding process: cell seeding efficiency (CSE) and cell-specific viability (CSV). In addition, cell spatial distribution (CSD) was analyzed by Live/Dead staining assays. Analysis identified a number of statistically significant main factor effects and interactions. Among the five seeding factors, only seeding volume and seeding time significantly affected CSE and CSV. Also, cell and scaffold type were involved in the interactions with other seeding factors. Within the investigated ranges, optimal conditions in terms of CSV and CSD were obtained when seeding cells in a regular scaffold with an excess of medium. The results of this case study contribute to a better understanding and definition of optimal process parameters for cell seeding. A DOE strategy can identify and optimize critical process variables to reduce the variability and assists in determining which variables should be carefully controlled during good manufacturing practice production to enable a clinically relevant implant.