RESUMEN
PCR has been a reliable and inexpensive method for nucleic acid detection in the past several decades. In particular, multiplex PCR is a powerful tool to analyze many biomarkers in the same reaction, thus maximizing detection sensitivity and reducing sample usage. However, balancing the amplification kinetics between amplicons and distinguishing them can be challenging, diminishing the broad adoption of high order multiplex PCR panels. Here, we present a new paradigm in PCR amplification and multiplexed detection using UltraPCR. UltraPCR utilizes a simple centrifugation workflow to split a PCR reaction into â¼34 million partitions, forming an optically clear pellet of spatially separated reaction compartments in a PCR tube. After in situ thermocycling, light sheet scanning is used to produce a 3D reconstruction of the fluorescent positive compartments within the pellet. At typical sample DNA concentrations, the magnitude of partitions offered by UltraPCR dictate that the vast majority of target molecules occupy a compartment uniquely. This single molecule realm allows for isolated amplification events, thereby eliminating competition between different targets and generating unambiguous optical signals for detection. Using a 4-color optical setup, we demonstrate that we can incorporate 10 different fluorescent dyes in the same UltraPCR reaction. We further push multiplexing to an unprecedented level by combinatorial labeling with fluorescent dyes - referred to as "comboplex" technology. Using the same 4-color optical setup, we developed a 22-target comboplex panel that can detect all targets simultaneously at high precision. Collectively, UltraPCR has the potential to push PCR applications beyond what is currently available, enabling a new class of precision genomics assays.
RESUMEN
Digital PCR (dPCR) was first conceived for single-molecule quantitation. However, current dPCR systems often require DNA templates to share partitions due to limited partitioning capacities. Here, we introduce UltraPCR, a next-generation dPCR system where DNA counting is performed in a single-molecule regimen through a 6-log dynamic range using a swift and parallelized workflow. Each UltraPCR reaction is divided into >30 million partitions without microfluidics to achieve single template occupancy. Combined with a unique emulsion chemistry, partitions are optically clear, enabling the use of a three-dimensional imaging technique to rapidly detect DNA-positive partitions. Single-molecule occupancy also allows for more straightforward multiplex assay development due to the absence of partition-specific competition. As a proof of concept, we developed a 222-plex UltraPCR assay and demonstrated its potential use as a rapid, low-cost screening assay for noninvasive prenatal testing for as low as 4% trisomy fraction samples with high precision, accuracy, and reproducibility.
Asunto(s)
ADN , Pruebas Prenatales no Invasivas , Embarazo , Femenino , Humanos , Reproducibilidad de los Resultados , ADN/química , Reacción en Cadena de la Polimerasa/métodos , Replicación del ADNRESUMEN
Cell-based therapies offer great promise for repairing cartilage. Previous strategies often involved using a single cell population such as stem cells or chondrocytes. A mixed cell population may offer an alternative strategy for cartilage regeneration while overcoming donor scarcity. We have recently reported that adipose-derived stem cells (ADSCs) can catalyze neocartilage formation by neonatal chondrocytes (NChons) when mixed co-cultured in 3D hydrogels in vitro. However, it remains unknown how the biochemical and mechanical cues of hydrogels modulate cartilage formation by mixed cell populations in vivo. The present study seeks to answer this question by co-encapsulating ADSCs and NChons in 3D hydrogels with tunable stiffness (â¼1-33 kPa) and biochemical cues, and evaluating cartilage formation in vivo using a mouse subcutaneous model. Three extracellular matrix molecules were examined, including chondroitin sulfate (CS), hyaluronic acid (HA), and heparan sulfate (HS). Our results showed that the type of biochemical cue played a dominant role in modulating neocartilage deposition. CS and HA enhanced type II collagen deposition, a desirable phenotype for articular cartilage. In contrast, HS promoted fibrocartilage phenotype with the upregulation of type I collagen and failed to retain newly deposited matrix. Hydrogels with stiffnesses of â¼7-33 kPa led to a comparable degree of neocartilage formation, and a minimal initial stiffness was required to retain hydrogel integrity over time. Results from this study highlight the important role of matrix cues in directing neocartilage formation, and they offer valuable insights in guiding optimal scaffold design for cartilage regeneration by using mixed cell populations.
Asunto(s)
Cartílago , Células Inmovilizadas , Condrocitos/metabolismo , Hidrogeles , Regeneración , Células Madre/metabolismo , Animales , Cartílago/lesiones , Cartílago/fisiología , Células Inmovilizadas/metabolismo , Células Inmovilizadas/trasplante , Fuerza Compresiva , Xenoinjertos , Humanos , Hidrogeles/química , Hidrogeles/farmacología , Ratones DesnudosRESUMEN
Despite increasing evidence that meniscal degeneration is an early event in the development of knee osteoarthritis, relatively little is known regarding the sequence or functional implications of cytokine-induced meniscal degradation or how degradation varies with age. This study examined dose-dependent patterns of interleukin-1 (IL-1)-induced matrix degradation in explants from the radially middle regions of juvenile and adult bovine menisci. Tissue explants were cultured for 10 days in the presence of 0, 1.25, 5, or 20 ng/ml recombinant human IL-1α. Juvenile explants exhibited immediate and extensive sulfated glycosaminoglycan (sGAG) loss and subsequent collagen release beginning after 4-6 days, with relatively little IL-1 dose-dependence. Adult explants exhibited a more graded response to IL-1, with dose-dependent sGAG release and a lower fraction of sGAG released (but greater absolute release) than juvenile explants. In contrast to juvenile explants, adult explants exhibited minimal collagen release over the 10-day culture. Compressive and shear moduli reflected the changes in explant composition, with substantial decreases for both ages but a greater relative decrease in juvenile tissue. Dynamic moduli exhibited stronger dependence on explant sGAG content for juvenile tissue, likely reflecting concomitant changes to both proteoglycan and collagen tissue components. The patterns of tissue degradation suggest that, like in articular cartilage, meniscal proteoglycans may partially protect collagen from cell-mediated degeneration. A more detailed view of functional changes in meniscal tissue mechanics with degeneration will help to establish the relevance of in vitro culture models and will advance understanding of how meniscal degeneration contributes to overall joint changes in early stage osteoarthritis. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:801-811, 2016.
Asunto(s)
Menisco/patología , Osteoartritis/etiología , Animales , Bovinos , Interleucina-1 , Osteoartritis/patologíaRESUMEN
Stem cells can contribute to cartilage repair either directly through chondrogenic differentiation or indirectly through paracrine signaling. Using a 3D co-culture model, we have recently reported that adipose-derived stem cells (ADSCs) can catalyze cartilage formation by neonatal chondrocytes (NChons) when mixed co-cultured in biomimetic hydrogels. However, how matrix cues influence such catalyzed cartilage formation remains unknown. To answer this question, ADSCs and NChons were co-encapsulated in 39 combinatorial hydrogel compositions with decoupled biochemical and mechanical properties. Methacrylated extracellular matrix (ECM) molecules including chondroitin sulfate, hyaluronic acid and heparan sulfate were incorporated at varying concentrations (0.5%, 1.25%, 2.5% and 5%) (w/v). Mechanical testing confirmed that hydrogel stiffness was largely decoupled from ECM cues (15 kPa, 40 kPa and 100 kPa). The biochemical assay and histology results showed that the type of ECM cue played a dominant role in modulating catalyzed cartilage formation, while varying hydrogel stiffness and doses of ECM led to more modest changes. Both chondroitin sulfate and hyaluronic acid led to robust articular cartilage matrix deposition, as shown by the intense staining of aggrecan and type II collagen. In soft hydrogels (15 kPa), chondroitin sulfate led to the highest amount of sulfated glycosaminoglycan deposition and increased compressive moduli. In contrast, heparan sulfate promoted type I collagen deposition, an undesirable fibrocartilage phenotype, and increasing heparan sulfate decreased cell proliferation and ECM deposition. Findings from the present study may guide the optimal scaffold design to maximize the synergistic cartilage formation using mixed cell populations.
RESUMEN
Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.
Asunto(s)
Cartílago Articular/citología , Condrocitos/citología , Técnicas Citológicas/métodos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Andamios del Tejido , Adolescente , Adulto , Factores de Edad , Materiales Biomiméticos/química , Sulfatos de Condroitina/química , Humanos , Polietilenglicoles/química , Regeneración/fisiología , Cicatrización de HeridasRESUMEN
Stem cells hold great promise for treating cartilage degenerative diseases such as osteoarthritis (OA). The efficacy of stem cell-based therapy for cartilage repair is highly dependent on their interactions with local cells in the joint. This study aims at evaluating the interactions between osteoarthritic chondrocytes (OACs) and adipose-derived stem cells (ADSCs) using three dimensional (3D) biomimetic hydrogels. To examine the effects of cell distribution on such interactions, ADSCs and OACs were co-cultured in 3D using three co-culture models: conditioned medium (CM), bi-layered, and mixed co-culture with varying cell ratios. Furthermore, the effect of transforming growth factor (TGF)-ß3 supplementation on ADSC-OAC interactions and the resulting cartilage formation was examined. Outcomes were analyzed using quantitative gene expression, cell proliferation, cartilage matrix production, and histology. TGF-ß3 supplementation led to a substantial increase in cartilage matrix depositions in all groups, but had differential effects on OAC-ADSC interactions in different co-culture models. In the absence of TGF-ß3, CM or bi-layered co-culture had negligible effects on gene expression or cartilage formation. With TGF-ß3 supplementation, CM and bi-layered co-culture inhibited cartilage formation by both ADSCs and OACs. In contrast, a mixed co-culture with moderate OAC ratios (25% and 50%) resulted in synergistic interactions with enhanced cartilage matrix deposition and reduced catabolic marker expression. Our results suggested that the interaction between OACs and ADSCs is highly dependent on cell distribution in 3D and soluble factors, which should be taken into consideration when designing stem cell-based therapy for treating OA patients.
Asunto(s)
Tejido Adiposo/citología , Comunicación Celular/efectos de los fármacos , Condrocitos/patología , Osteoartritis/patología , Células Madre/citología , Factor de Crecimiento Transformador beta3/farmacología , Anciano , Biomarcadores/metabolismo , Cartílago/efectos de los fármacos , Cartílago/crecimiento & desarrollo , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Condrogénesis/efectos de los fármacos , Técnicas de Cocultivo , Colágeno Tipo II/metabolismo , Medios de Cultivo Condicionados/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Persona de Mediana Edad , Células Madre/efectos de los fármacos , Células Madre/metabolismoRESUMEN
Regeneration of human articular cartilage is inherently limited and extensive efforts have focused on engineering the cartilage tissue. Various cellular sources have been studied for cartilage tissue engineering including adult chondrocytes, and embryonic or adult stem cells. Juvenile chondrocytes (from donors below 13 years of age) have recently been reported to be a promising cell source for cartilage regeneration. Previous studies have compared the potential of adult and juvenile chondrocytes or adult and osteoarthritic (OA) chondrocytes. To comprehensively characterize the comparative potential of young, old, and diseased chondrocytes, here we examined cartilage formation by juvenile, adult, and OA chondrocytes in three-dimensional (3D) biomimetic hydrogels composed of poly(ethylene glycol) and chondroitin sulfate. All three human articular chondrocytes were encapsulated in the 3D biomimetic hydrogels and cultured for 3 or 6 weeks to allow maturation and extracellular matrix formation. Outcomes were analyzed using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. After 3 and 6 weeks, juvenile chondrocytes showed a greater upregulation of chondrogenic gene expression than adult chondrocytes, while OA chondrocytes showed a downregulation. Aggrecan and type II collagen deposition and glycosaminoglycan accumulation were high for juvenile and adult chondrocytes but not for OA chondrocytes. Similar trend was observed in the compressive moduli of the cartilage constructs generated by the three different chondrocytes. In conclusion, the juvenile, adult and OA chondrocytes showed differential responses in the 3D biomimetic hydrogels. The 3D culture model described here may also provide a useful tool to further study the molecular differences among chondrocytes from different stages, which can help elucidate the mechanisms for age-related decline in the intrinsic capacity for cartilage repair.
Asunto(s)
Materiales Biomiméticos/farmacología , Cartílago Articular/citología , Condrocitos/citología , Condrogénesis , Hidrogeles/farmacología , Adolescente , Adulto , Biomarcadores/metabolismo , Fenómenos Biomecánicos/efectos de los fármacos , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrogénesis/efectos de los fármacos , Glicosaminoglicanos/metabolismo , Humanos , Masculino , Osteoartritis/patología , Andamios del TejidoRESUMEN
Regeneration of human cartilage is inherently inefficient. Current cell-based approaches for cartilage repair, including autologous chondrocytes, are limited by the paucity of cells, associated donor site morbidity, and generation of functionally inferior fibrocartilage rather than articular cartilage. Upon investigating the role of collagen VI (Col VI), a major component of the chondrocyte pericellular matrix (PCM), we observe that soluble Col VI stimulates chondrocyte proliferation. Interestingly, both adult and osteoarthritis chondrocytes respond to soluble Col VI in a similar manner. The proliferative effect is, however, strictly due to the soluble Col VI as no proliferation is observed upon exposure of chondrocytes to immobilized Col VI. Upon short Col VI treatment in 2D monolayer culture, chondrocytes maintain high expression of characteristic chondrocyte markers like Col2a1, agc, and Sox9 whereas the expression of the fibrocartilage marker Collagen I (Col I) and of the hypertrophy marker Collagen X (Col X) is minimal. Additionally, Col VI-expanded chondrocytes show a similar potential to untreated chondrocytes in engineering cartilage in 3D biomimetic hydrogel constructs. Our study has, therefore, identified soluble Col VI as a biologic that can be useful for the expansion and utilization of scarce sources of chondrocytes, potentially for autologous chondrocyte implantation. Additionally, our results underscore the importance of further investigating the changes in chondrocyte PCM with age and disease and the subsequent effects on chondrocyte growth and function.
Asunto(s)
Condrocitos/fisiología , Colágeno Tipo VI/farmacología , Ingeniería de Tejidos/métodos , Adulto , Animales , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Condrocitos/citología , Condrocitos/efectos de los fármacos , Humanos , MasculinoRESUMEN
Adipose-derived stromal cells (ADSCs) are attractive autologous cell sources for cartilage repair given their relative abundance and ease of isolation. Previous studies have demonstrated the potential of extracellular matrix (ECM) molecules as three-dimensional (3D) scaffolds for promoting chondrogenesis. However, few studies have compared the effects of varying types or doses of ECM molecules on chondrogenesis of ADSCs in 3D. Furthermore, increasing ECM molecule concentrations often result in simultaneous changes in the matrix stiffness, which makes it difficult to elucidate the relative contribution of biochemical cues or matrix stiffness on stem cell fate. Here we report the development of an ECM-containing hydrogel platform with largely decoupled biochemical and mechanical cues by modulating the degree of methacrylation of ECM molecules. Specifically, we incorporated three types of ECM molecules that are commonly found in the cartilage matrix, including chondroitin sulfate (CS), hyaluronic acid (HA), and heparan sulfate (HS). To elucidate the effects of interactive biochemical and mechanical signaling on chondrogenesis, ADSCs were encapsulated in 39 combinatorial hydrogel compositions with independently tunable ECM types (CS, HA, and HS), concentrations (0.5%, 1.25%, 2.5%, and 5% [w/v]), and matrix stiffness (3, 30, and 90 kPa). Our results show that the effect of ECM composition on chondrogenesis is dependent on the matrix stiffness of hydrogels, suggesting that matrix stiffness and biochemical cues interact in a nonlinear manner to regulate chondrogenesis of ADSCs in 3D. In soft hydrogels (~3 kPa), increasing HA concentrations resulted in substantial upregulation of aggrecan and collagen type II expression in a dose-dependent manner. This trend was reversed in HA-containing hydrogels with higher stiffness (~90 kPa). The platform reported herein could provide a useful tool for elucidating how ECM biochemical cues and matrix stiffness interact together to regulate stem cell fate, and for rapidly optimizing ECM-containing scaffolds to support stem cell differentiation and tissue regeneration.
Asunto(s)
Tejido Adiposo/citología , Diferenciación Celular/efectos de los fármacos , Condrogénesis/efectos de los fármacos , Hidrogeles/farmacología , Proteínas Matrilinas/metabolismo , Fenómenos Biomecánicos/efectos de los fármacos , Diferenciación Celular/genética , Condrogénesis/genética , Módulo de Elasticidad/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismoRESUMEN
Cartilage loss is a leading cause of disability among adults and effective therapy remains elusive. Neonatal chondrocytes (NChons) are an attractive allogeneic cell source for cartilage repair, but their clinical translation has been hindered by scarce donor availability. Here we examine the potential for catalyzing cartilage tissue formation using a minimal number of NChons by co-culturing them with adipose-derived stem cells (ADSCs) in 3D hydrogels. Using three different co-culture models, we demonstrated that the effects of co-culture on cartilage tissue formation are dependent on the intercellular distance and cell distribution in 3D. Unexpectedly, increasing ADSC ratio in mixed co-culture led to increased synergy between NChons and ADSCs, and resulted in the formation of large neocartilage nodules. This work raises the potential of utilizing stem cells to catalyze tissue formation by neonatal chondrocytes via paracrine signaling, and highlights the importance of controlling cell distribution in 3D matrices to achieve optimal synergy.
Asunto(s)
Materiales Biomiméticos/farmacología , Cartílago Articular/citología , Condrocitos/metabolismo , Hidrogeles/farmacología , Células Madre Mesenquimatosas/metabolismo , Tejido Adiposo/citología , Animales , Cartílago Articular/patología , Bovinos , Proliferación Celular , Células Cultivadas , Condrocitos/citología , Condrogénesis , Técnicas de Cocultivo , Perfilación de la Expresión Génica , Regeneración Tisular Dirigida , Humanos , Células Madre Mesenquimatosas/citología , Comunicación Paracrina , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
Macropores in tissue engineering scaffolds provide space for vascularization, cell-proliferation and cellular interactions, and is crucial for successful tissue regeneration. Modulating the size and density of macropores may promote desirable cellular processes at different stages of tissue development. Most current techniques for fabricating macroporous scaffolds produce fixed macroporosity and do not allow the control of porosity during cell culture. Most macropore-forming techniques also involve non-physiological conditions, such that cells can only be seeded in a post-fabrication process, which often leads to low cell seeding efficiency and uneven cell distribution. Here we report a process to create dynamic hydrogels as tissue engineering scaffolds with tunable macroporosity using stimuli-responsive porogens of gelatin, alginate and hyaluronic acid, which degrade in response to specific stimuli including temperature, chelating and enzymatic digestion, respectively. SEM imaging confirmed sequential pore formation in response to sequential stimulations: 37 °C on day 0, EDTA on day 7, and hyaluronidase on day 14. Bovine chondrocytes were encapsulated in the Alg porogen, which served as cell-delivery vehicles, and changes in cell viability, proliferation and tissue formation during sequential stimuli treatments were evaluated. Our results showed effective cell release from Alg porogen with high cell viability and markedly increased cell proliferation and spreading throughout the 3D hydrogels. Dynamic pore formation also led to significantly enhanced type II and X collagen production by chondrocytes. This platform provides a valuable tool to create stimuli-responsive scaffolds with dynamic macroporosity for a broad range of tissue engineering applications, and may also be used for fundamental studies to examine cell responses to dynamic niche properties.
Asunto(s)
Hidrogeles/farmacología , Ingeniería de Tejidos , Andamios del Tejido/química , Alginatos/farmacología , Animales , Bovinos , Recuento de Células , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Condrocitos/citología , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Técnica del Anticuerpo Fluorescente , Gelatina/química , Gelatina/farmacología , Ácido Glucurónico/farmacología , Ácidos Hexurónicos/farmacología , Ácido Hialurónico/química , Ácido Hialurónico/farmacología , Microscopía Electrónica de Rastreo , PorosidadRESUMEN
Stem cells reside in a multi-factorial environment containing biochemical and mechanical signals. Changing biochemical signals in most scaffolds often leads to simultaneous changes in mechanical properties, which makes it difficult to elucidate the complex interplay between niche cues. Combinatorial studies on cell-material interactions have emerged as a tool to facilitate analyses of stem cell responses to various niche cues, but most studies to date have been performed on two-dimensional environments. Here we developed three-dimensional combinatorial hydrogels with independent control of biochemical and mechanical properties to facilitate analysis of interactive biochemical and mechanical signaling on adipose-derived stem cell osteogenesis in three dimensions. Our results suggest that scaffold biochemical and mechanical signals synergize only at specific combinations to promote bone differentiation. Leading compositions were identified to have intermediate stiffness (â¼55kPa) and low concentration of fibronectin (10µg ml(-1)), which led to an increase in osteocalcin gene expression of over 130-fold. Our results suggest that scaffolds with independently tunable niche cues could provide a powerful tool for conducting mechanistic studies to decipher how complex niche cues regulate stem cell fate in three dimensions, and facilitate rapid identification of optimal niche cues that promote desirable cellular processes or tissue regeneration.
Asunto(s)
Tejido Adiposo/citología , Diferenciación Celular/efectos de los fármacos , Hidrogeles/farmacología , Fenómenos Mecánicos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Células Madre/metabolismo , Antraquinonas/metabolismo , Biomarcadores/metabolismo , Calcio/metabolismo , Supervivencia Celular/efectos de los fármacos , Colágeno Tipo I/metabolismo , Colágeno Tipo II/metabolismo , Fuerza Compresiva/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Miosina Tipo IIB no Muscular/genética , Miosina Tipo IIB no Muscular/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Coloración y Etiquetado , Células Madre/citología , Células Madre/efectos de los fármacosRESUMEN
Despite over two decades of research on cartilage tissue engineering, very few products have moved from bench to bedside and effective therapy remains lacking. This review discusses recent progress in developing novel strategies for engineering cartilage tissues with long-term functionality. Specifically we focus on the following aspects including identifying promising cell sources, designing 3D scaffolds with dynamic and spatially patterned cues to guide desired cellular processes, mimicking zonal organization, integrating with host tissue, and monitoring cell fate and tissue regeneration in situ.
Asunto(s)
Cartílago/citología , Ingeniería de Tejidos/tendencias , Animales , Cartílago/fisiología , Diferenciación Celular , Fenómenos Fisiológicos Celulares , Microambiente Celular , Humanos , RegeneraciónRESUMEN
Research on the adhesive locomotion of terrestrial gastropods is gaining renewed interest as it provides a source of guidance for the design of soft biomimetic robots that can perform functions currently not achievable by conventional rigid vehicles. The locomotion of terrestrial gastropods is driven by a train of periodic muscle contractions (pedal waves) and relaxations (interwaves) that propagate from their tails to their heads. These ventral waves interact with a thin layer of mucus secreted by the animal that transmits propulsive forces to the ground. The exact mechanism by which these propulsive forces are generated is still a matter of controversy. Specifically, the exact role played by the complex rheological and adhesive properties of the mucus is not clear. To provide quantitative data that could shed light on this question, we use a newly developed technique to measure, with high temporal and spatial resolution, the propulsive forces that terrestrial gastropods generate while crawling on smooth flat surfaces. The traction force measurements demonstrate the importance of the finite yield stress of the mucus in generating thrust and are consistent with the surface of the ventral foot being lifted with the passage of each pedal wave. We also show that a forward propulsive force is generated beneath each stationary interwave and that this net forward component is balanced by the resistance caused by the outer rim of the ventral foot, which slides at the speed of the center of mass of the animal. Simultaneously, the animal pulls the rim laterally inward. Analysis of the traction forces reveals that the kinematics of the pedal waves is far more complex than previously thought, showing significant spatial variation (acceleration/deceleration) as the waves move from the tail to the head of the animal.