Characteristics of cartilage engineered from human pediatric auricular cartilage

In the repair of cartilage defects, autologous tissue offers the advantage of lasting biocompatibility. The ability of bovine chondrocytes isolated from hyaline cartilage to generate tissue-engineered cartilage in a predetermined shape, such as a human ear, has been demonstrated; however, the potential of chondrocytes isolated from human elastic cartilage remains unknown. In this study, the authors examined the multiplication characteristics of human auricular chondrocytes and the ability of these cells to generate new elastic cartilage as a function of the length of time they are maintained in vitro. Human auricular cartilage, harvested from patients 5 to 17 years of age, was digested in collagenase, and the chondrocytes were isolated and cultured in vitro for up to 12 weeks. Cells were trypsinized, counted, and passaged every 2 weeks. Chondrocyte-polymer (polyglycolic acid) constructs were created at each passage and then implanted into athymic mice for 8 weeks. The ability of the cells to multiply in vitro and their ability to generate new cartilage as a function of the time they had been maintained in vitro were studied. A total of 31 experimental constructs from 12 patients were implanted and compared with a control group of constructs without chondrocytes. In parallel, a representative sample of cells was evaluated to determine the presence of collagen. The doubling rate of human auricular chondrocytes in vitro remained constant within the population studied. New tissue developed in 22 of 31 experimental implants. This tissue demonstrated the physical characteristics of auricular cartilage on gross inspection. Histologically, specimens exhibited dense cellularity and lacunae-containing cells embedded in a basophilic matrix. The specimens resembled immature cartilage and were partially devoid of the synthetic material of which the construct had been composed. Analyses for collagen, proteoglycans, and elastin were consistent with elastic cartilage. No cartilage was detected in the control implants. Human auricular chondrocytes multiply well in vitro and possess the ability to form new cartilage when seeded onto a three-dimensional scaffold. These growth characteristics might some day enable chondrocytes isolated from a small auricular biopsy to be expanded in vitro to generate a large, custom-shaped, autologous graft for clinical reconstruction of a cartilage defect, such as for congenital microtia.     

Impacts of engineered nanomaterials on microbial community structure and function in natural and engineered ecosystems

In natural and engineered environments, microorganisms often exist as complex communities, which are key to the health of ecosystems and the success of bioprocesses in various engineering applications. With the rapid development of nanotechnology in recent years, engineered nanomaterials (ENMs) have been considered one type of emerging contaminants that pose great potential risks to the proper function of microbial communities in natural and engineered ecosystems. The impacts of ENMs on microorganisms have attracted increasing research attentions; however, most studies focused on the antimicrobial activities of ENMs at single cell and population level. Elucidating the influence of ENMs on microbial communities represents a critical step toward a comprehensive understanding of the ecotoxicity of ENMs. In this mini-review, we summarize and discuss recent research work on the impacts of ENMs on microbial communities in natural and engineered ecosystems, with an emphasis on their influences on the community structure and function. We also highlight several important research topics which may be of great interest to the research community.     

A novel lake-zoning framework for large lakes based on numerical modelling

Because of the high spatial heterogeneity of the ecosystem properties of large lakes, the characterisation of lake zones is particularly helpful for designing zone-specific strategies for water management practices. In this study, we developed a lake-zoning framework by investigating the lateral dispersion of inflow pollutants through dye-tracking experiments in a case study of a large shallow lake (Lake Chaohu) in China. A two-dimensional hydrodynamic and mass transport model was used to quantify the impacts of inflow rivers on this lake. Results from 2012 to 2014 showed that the lake zoning results were more stable at the annual scale than at the seasonal scale, implying that the seasonal scale is not suitable for the establishment of lake zones. A driving factor analysis showed that inflow discharge was a more critical factor determining lake zones than wind conditions. In contrast to previous lake zoning methods, the proposed framework implements lake zoning quantitatively through hydrodynamic modelling.     

Computational modeling of nutrient utilization in engineered cartilage

This study presents a mathematical model for simulating cartilaginous culture of chondrocytes seeded in scaffolds and for investigating the effects of glucose and oxygen concentration and pH value on cell metabolic rates. The model can clearly interpret the unexplained experimental observation (Sengers BG, Heywood HK, Lee DA, Oomens CWJ, Bader DL. Nutrient utilization by bovine articular chondrocytes: A combined experimental and theoretical approach. J Biomech Eng. 2005;127:758–766.), which showed that the oxygen concentration within the scaffold may increase instead of continuously decreasing in static cartilaginous culture of chondrocytes. Results from simulation demonstrate that when cells metabolize glucose and form lactate under high glucose concentration conditions, the acidity in the culture environment increases, inhibiting cell metabolic rates in the process. Consequently, the rate of oxygen consumption decreases in later stages of cell culture. As oxygen can be replenished through the free surface of the culture medium, oxygen concentration within the scaffold increases rather than decreases over time in the acidic environment. Different initial glucose concentration yields different results. In low glucose concentration conditions, oxygen concentration basically keeps decreasing with culture time. This is because the pH in the environment does not significantly change because of slower glycolysis rate in low glucose concentration cases, forming less lactic acid. From the simulation results, additional information regarding in vitro culture of chondrocytes is obtained. The correlations between nutrient consumption, lactate secretion, and pH changes during cell culture are also understood and may serve as a reference for in vitro cell culture research of tissue engineering. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 452–462, 2013     

Bioreactor studies of native and tissue engineered cartilage

Functional tissue engineering of cartilage involves the use of bioreactors designed to provide a controlled in vitro environment that embodies some of the biochemical and physical signals known to regulate chondrogenesis. Hydrodynamic conditions can affect in vitro tissue formation in at least two ways: by direct effects of hydrodynamic forces on cell morphology and function, and by indirect flow-induced changes in mass transfer of nutrients and metabolites. In the present work, we discuss the effects of three different in vitro environments: static flasks (tissues fixed in place, static medium), mixed flasks (tissues fixed in place, unidirectional turbulent flow) and rotating bioreactors (tissues dynamically suspended in laminar flow) on engineered cartilage constructs and native cartilage explants. As compared to static and mixed flasks, dynamic laminar flow in rotating bioreactors resulted in the most rapid tissue growth and the highest final fractions of glycosaminoglycans and total collagen in both tissues. Mechanical properties (equilibrium modulus, dynamic stiffness, hydraulic permeability) of engineered constructs and explanted cartilage correlated with the wet weight fractions of glycosaminoglycans and collagen. Current research needs in the area of cartilage tissue engineering include the utilization of additional physiologically relevant regulatory signals, and the development of predictive mathematical models that enable optimization of the conditions and duration of tissue culture.     

Tissue‐engineered cartilage of porcine and human origin as in vitro test system in arthritis research

The increasing prevalence of cartilage destruction during arthritis has entailed an intensified amount for in vitro cartilage models to analyze pathophysiological processes and to screen for antirheumatic drugs. Tissue engineering offers the opportunity to establish highly organized 3D cell cultures facilitating the formation of in vitro models that reflect the human situation. We report the comparison of porcine chondrocyte pellet and alginate bead cultures as model systems for human cartilage and the further development into a human system that was applied in an arthritis model. In porcine pellet and alginate cultures, formation of cartilage matrix similar to human matrix was verified by histology and PCR. As alginate beads could be cultivated batch‐wise in one well of a multiwell plate, we further developed this setting into a human system. In contrast, each pellet had to be cultivated individually in one well of a multiwell plate, which is time consuming. Following stimulation of human chondrocyte alginate cultures with conditioned media from human synovial fibroblasts derived from arthritis patients, microarray analysis verified the induction of genes related to cartilage destruction (like MMP10, ?12) and inflammation (like IL6, ?8 and chemokines). Several genes are coding for proteins that are members of inflammatory and catabolic pathways. Belonging to the most affected pathways, we identified the focal adhesion, cytokine–cytokine receptor interaction, ECM‐receptor signalling, Jak‐STAT signalling, and toll‐like receptor signalling pathways, all relevant in arthritis. Therefore, we demonstrate that engineered cartilage of porcine and human origin represents a powerful in vitro model for cartilage in vivo. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Functional properties of bone marrow-derived MSC-based engineered cartilage are unstable with very long-term in vitro culture

The success of stem cell-based cartilage repair requires that the regenerate tissue reach a stable state. To investigate the long-term stability of tissue engineered cartilage constructs, we assessed the development of compressive mechanical properties of chondrocyte and mesenchymal stem cell (MSC)-laden three dimensional agarose constructs cultured in a well defined chondrogenic in vitro environment through 112 days. Consistent with previous reports, in the presence of TGF-β, chondrocytes outperformed MSCs through day 56, under both free swelling and dynamic culture conditions, with MSC-laden constructs reaching a plateau in mechanical properties between days 28 and 56. Extending cultures through day 112 revealed that MSCs did not simply experience a lag in chondrogenesis, but rather that construct mechanical properties never matched those of chondrocyte-laden constructs. After 56 days, MSC-laden constructs underwent a marked reversal in their growth trajectory, with significant declines in glycosaminoglycan content and mechanical properties. Quantification of viability showed marked differences in cell health between chondrocytes and MSCs throughout the culture period, with MSC-laden construct cell viability falling to very low levels at these extended time points. These results were not dependent on the material environment, as similar findings were observed in a photocrosslinkable hyaluronic acid (HA) hydrogel system that is highly supportive of MSC chondrogenesis. These data suggest that, even within a controlled in vitro environment that is conducive to chondrogenesis, there may be an innate instability in the MSC phenotype that is independent of scaffold composition, and may ultimately limit their application in functional cartilage repair.     

Modeling the dynamic composition of engineered cartilage

Mathematical models to describe extracellular matrix (ECM) deposition and scaffold degradation in cell-polymer constructs for the design of engineered cartilage were developed and validated. The ECM deposition model characterized a product-inhibition mechanism in the concentration of cartilage molecules, collagen and glycosaminoglycans (GAG). The scaffold degradation model used first-order kinetics to describe hydrolysis (not limited by diffusion) of biodegradable polyesters, polyglycolic acid and polylactic acid. Each model was fit to published accumulation and degradation data. As experimental validation, cell-polymer constructs (n=24) and unseeded scaffolds (n=24) were cultured in vitro. Biochemical assays for ECM content and measurements of scaffold mass were performed at 1, 2, 4, 6, 8, or 10 weeks (n=8 per time point). The models demonstrated a strong fit with published data and experimental results (R(2)=0.75 to 0.99) and predicted the temporal total construct mass with reasonable accuracy (30% RMS error). This approach can elucidate mechanisms governing accumulation/degradation and may be coupled with structure-function relationships to describe time-dependent changes in construct elastic properties.     

Engineering superficial zone features in tissue engineered cartilage

A major challenge in cartilage tissue engineering is the need to recreate the native tissue's anisotropic extracellular matrix structure. This anisotropy has important mechanical and biological consequences and could be crucial for integrative repair. Here, we report that hydrodynamic conditions that mimic the motion‐induced flow fields in between the articular surfaces in the synovial joint induce the formation of a distinct superficial layer in tissue engineered cartilage hydrogels, with enhanced production of cartilage matrix proteoglycan and Type II collagen. Moreover, the flow stimulation at the surface induces the production of the surface zone protein Proteoglycan 4 (aka PRG4 or lubricin). Analysis of second harmonic generation signature of collagen in this superficial layer reveals a highly aligned fibrillar matrix that resembles the alignment pattern in native tissue's surface zone, suggesting that mimicking synovial fluid flow at the cartilage surface in hydrodynamic bioreactors could be key to creating engineered cartilage with superficial zone features. Biotechnol. Bioeng. 2013; 110: 1476–1486. © 2012 Wiley Periodicals, Inc.     

Lumican inhibits collagen deposition in tissue engineered cartilage

Lumican is a glycoprotein that is found in the extracellular matrix of many connective tissues, including cartilage. It is a member of the small leucine-rich repeat proteoglycans family and along with two others, decorin and fibromodulin, has the capacity to bind to fibrillar collagens and limit their growth. Cartilage tissue engineering provides a potential method for the production of three-dimensional tissue for implantation into eroded joints. Many studies have demonstrated the growth of cartilage in vitro. However in all cases, biochemical analysis of the tissue revealed a significant deficit in the collagen content. We have now tested the hypothesis that the reduced collagen accumulation in engineered cartilage is a result of over-expression of decorin, fibromodulin or lumican. We have found that the lumican gene and protein are both over-expressed in engineered compared to natural cartilage whereas this is not the case for decorin or fibromodulin. Using a small hairpin lumican antisense sequence we were able to knockdown the lumican gene and protein expression in chondrocytes being used for tissue engineering. This resulted in increased accumulation of type II collagen (the major collagen of cartilage) whilst there was no significant alteration in the proteoglycan content. Furthermore, the antisense knockdown of lumican resulted in an increase in the average collagen fibril diameter measured by transmission electron microscopy. These results suggest that lumican plays a pivotal role in the development of tissue engineered cartilage and that regulation of this protein may be important for the production of high-quality implants.     

Hydrodynamics of vertical subsurface flow constructed wetlands: Tracer tests with rhodamine WT and numerical modelling

Typical unsteady unsaturated conditions can profoundly affect the hydrodynamics of vertical subsurface flow constructed wetlands. In this study we analyzed the hydrodynamics of a 33 m² vertical flow pilot plant, treating municipal secondary effluents. Three different saturation conditions were analyzed under several constant flux regimes: complete saturation, partial saturation with the free water table 20 cm over the bottom of the bed, and complete drainage. Tracer tests were performed in steady state conditions by dosing rhodamine WT as square input signals. Breakthrough curves were analyzed by means of both a classical residence time distribution analysis and an originally developed numerical plug-flow model with longitudinal dispersion adapted to the unsaturated conditions. We found that the degree of global mixing in the vertical flow constructed wetland increased as the water content increased; this effect was controlled by the hydraulic residence time of the system. Conversely, the degree of local mixing was inversely affected by water content; the dispersivity was 4.5, 10, and 14 cm for fully saturated, partially saturated and draining conditions, respectively. We explain the dependency of dispersivity on water content in physical terms; however, further studies are needed to mathematically include this relationship in numerical models that describe the behaviour of vertical flow constructed wetlands.     

Systematic in vitro specificity profiling reveals nicking defects in natural and engineered CRISPR–Cas9 variants

Cas9 is an RNA-guided endonuclease in the bacterial CRISPR–Cas immune system and a popular tool for genome editing. The commonly used Streptococcus pyogenes Cas9 (SpCas9) is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific. However, previous studies have focused on specificity of double-strand break (DSB) or indel formation, potentially overlooking alternative cleavage activities of these Cas9 variants. In this study, we employed in vitro cleavage assays of target libraries coupled with high-throughput sequencing to systematically compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9 and HiFi Cas9). We observed that all Cas9s tested could cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on target sequence and Cas9 variant. In addition, SaCas9 and engineered SpCas9 variants nick targets with multiple mismatches but have a defect in generating a DSB, while SpCas9 creates DSBs at these targets. Overall, these differences in cleavage rates and DSB formation may contribute to varied specificities observed in genome editing studies.     

Multiscale modelling for investigating the long-term time-dependent biphasic behaviour of the articular cartilage in the natural hip joint

Biomechanics and Modeling in Mechanobiology - A better understanding of the time-dependent biomechanical behaviour of the biphasic hip articular cartilage (AC) under physiological loadings is...     

Modulation of the mechanical properties of tissue engineered cartilage

Cartilaginous constructs have been grown in vitro using chondrocytes, biodegradable polymer scaffolds, and tissue culture bioreactors. In the present work, we studied how the composition and mechanical properties of engineered cartilage can be modulated by the conditions and duration of in vitro cultivation, using three different environments: static flasks, mixed flasks, and rotating vessels. After 4-6 weeks, static culture yielded small and fragile constructs, while turbulent flow in mixed flasks induced the formation of an outer fibrous capsule; both environments resulted in constructs with poor mechanical properties. The constructs that were cultured freely suspended in a dynamic laminar flow field in rotating vessels had the highest fractions of glycosaminoglycans and collagen (respectively 75% and 39% of levels measured in native cartilage), and the best mechanical properties (equilibrium modulus, hydraulic permeability, dynamic stiffness, and streaming potential were all about 20% of values measured in native cartilage). Chondrocytes in cartilaginous constructs remained metabolically active and phenotypically stable over prolonged cultivation in rotating bioreactors. The wet weight fraction of glycosaminoglycans and equilibrium modulus of 7 month constructs reached or exceeded the corresponding values measured from freshly explanted native cartilage. Taken together, these findings suggest that functional equivalents of native cartilage can be engineered by optimizing the hydrodynamic conditions in tissue culture bioreactors and the duration of tissue cultivation.     

Bioflocculation in natural and engineered systems: current perspectives

Microorganisms have the tendency to accumulate at interfaces through the release of extracellular polymeric substances to form aggregates such as films or flocs. This physical association leads to different modes of interactions among cells and the subsequent development of functionally and metabolically diverse consortia. Aggregation of cells in aqueous suspensions often results in the formation of flocs, which are hotspots of enhanced microbial processes. This has important implications for the dynamics of organic and inorganic matter in varied ecosystems. These microbial flocs are not only important components in nutrient turnover, decomposition, and sinking flux but also facilitate contaminant removal and treatment of wastewater and biomass harvesting. Greater insight into the multitude of interactions between microorganisms in flocs would be useful to enhance the efficiency of bioflocculation processes. This review covers the fundamental aspects and outlines the role of bioflocculation in controlled industrial processes and in nature.     

A mechanical composite spheres analysis of engineered cartilage dynamics

In the preparation of bioengineered reparative strategies for damaged or diseased tissues, the processes of biomaterial degradation and neotissue synthesis combine to affect the developing mechanical state of multiphase, composite engineered tissues. Here, cell-polymer constructs for engineered cartilage have been fabricated by seeding chondrocytes within three-dimensional scaffolds of biodegradable polymers. During culture, synthetic scaffolds degraded passively as the cells assembled an extracellular matrix (ECM) composed primarily of glycosaminoglycan and collagen. Biochemical and biomechanical assessment of the composite (cells, ECM, and polymer scaffold) were modeled at a unit-cell level to mathematically solve stress-strain relationships and thus construct elastic properties (n=4 samples per seven time points). This approach employed a composite spheres, micromechanical analysis to determine bulk moduli of: (1) the cellular-ECM inclusion within the supporting scaffold structure; and (2) the cellular inclusion within its ECM. Results indicate a dependence of constituent volume fractions with culture time (p<0.05). Overall mean bulk moduli were variably influenced by culture, as noted for the cell-ECM inclusion (K(c-m)=29.7 kPa, p=0.1439), the cellular inclusion (K(c)=5.5 kPa, p=0.0067), and its surrounding ECM (K(m)=373.9 kPa, p=0.0748), as well as the overall engineered construct (K=165.0 kPa, p=0.6899). This analytical technique provides a framework to describe the time-dependent contribution of cells, accumulating ECM, and a degrading scaffold affecting bioengineered construct mechanical properties.