Extraction, separation and purification of fatty acid ethyl esters for biodiesel and DHA from Thraustochytrid biomass.

A novel approach for in situ transesterification, extraction, separation, and purification of fatty acid ethyl esters (FAEE) for biodiesel and docosahexaenoic acid (DHA) from Thraustochytrid biomass has been developed. The downstream processing of Thraustochytrids oil necessitates optimization, considering the higher content of polyunsaturated fatty acids (PUFA). While two-step methods are commonly employed for extracting and transesterifying oil from oleaginous microbes, this may result in oxidation/epoxidation of omega-3 oil due to prolonged exposure to heat and oxygen. To address this issue, a rapid single-step method was devised for in situ transesterification of Thraustochytrid oil. Through further process optimization, a 50% reduction in solvent requirement was achieved without significantly impacting fatty acid recovery or composition. Scale-up studies in a 4 L reactor demonstrated complete FAEE recovery (99.98% of total oil) from biomass, concurrently enhancing DHA yield from 16% to nearly 22%. The decolorization of FAEE oil with fuller's earth effectively removed impurities such as pigments, secondary metabolites, and waxes, resulting in a clear, shiny appearance. High-performance liquid chromatography (HPLC) analysis indicated that the eluted DHA was over 94.5% pure, as corroborated by GC-FID analysis.

Simultaneous production of high-value lipids in Schizochytrium sp. by synergism of chemical modulators.

The study focuses on the simultaneous improvement of biomass, lipid, and docosahexaenoic acid (DHA) productivities in a single reactor using modulator control strategies. The efficacy of three different biochemical modulators, sesamol (Ses), 6-benzylaminopurine (6-BAP), and ethylenediaminetetraacetic acid (EDTA), as potential stimulants in augmenting the biomass, lipid, and DHA production of Schizochytrium sp. MTCC 5890 was elucidated. After 48 h of cultivation, among tested modulators, the individual supplementation of 6-BAP and Ses showed improvement in biomass, lipid, and DHA accumulation by 28.2%, 56.1%, and 87.2% and 21.7%, 47.9%, and 91%, respectively, over the non-supplemented group. In addition, the cooperative effect of selected concentrations, i.e., 10 mgL-1 6-BAP and 200 mgL-1 Ses, further increased the productivities of biomass of 13.5 gL-1d-1 ± 0.66, lipid of 7.4 gL-1d-1 ± 0.69, and DHA of 3.2 gL-1d-1 ± 1.09 representing 8%, 39%, and 69% increase over the individual addition of 6-BAP or Ses, respectively, in batch culture. Supplementation with 6-BAP + Ses at 12 h of time point eventually increased the lipid yield to 15.6 ± 0.42 gL-1 from 7.88 ± 0.31 gL-1 (control) and DHA yield to 6.4 ± 0.11 gL-1 from 2.23 ± 0.09 gL-1 (control), respectively. Furthermore, the process was optimized in continuous culture supplemented with 6-BAP + Ses for enhanced productivities. Continuous culture resulted in maximum biomass (2.04 ± 1.12 gL-1 day-1), lipid (1.0 ± 0.73 gL-1 day-1), and DHA (0.386 ± 0.22 gL-1 day-1) productivities, which were higher as compared with the batch and fed-batch processes by 26 ± 1.21%, 22 ± 1.01%, and 21 ± 0.98% and 24 ± 0.45%, 16 ± 0.38%, and 14 ± 0.12%, respectively. This work represents the potential application of the combined effect of modulators for the simultaneous enhancement of biomass production and lipid and DHA productivities. KEY POINTS: • The cumulative study of 6-BAP and sesamol proved to be more efficient in the simultaneous production of biomass, lipid, and DHA in a single reactor. • Addition of a combination of 6-BAP + Ses remarkably increased the biomass, lipid, and DHA productivities in tandem in continuous culture.

Morphologically favorable mutant of Trichoderma reesei for low viscosity cellulase production.

Metabolite production by filamentous fungi hampered because of high viscosity generated during growth. Low viscosity fermentation by mold is one of the preferred ways of large scale enzymes production. Cellulolytic enzymes play a key role during the process of lignocellulosic biomass conversion. In this study, a mutant RC-23-1 was isolated through mutagenesis (diethyl sulfate followed by UV) of Trichoderma reesei RUT-C30. RCRC-23-1 not only gave higher cellulase production but also generated lower viscosity during enzyme production. Viscosity of mutant growth was more than three times lower than parent strain. RC-23-1 shows unique, yeast-like colony morphology on solid media and small pellet-like growth in liquid media. This mutant did not spread like mold on solid media. This mutant produces cellulases constitutively when grown in sugars. Using only glucose, the cellulase production was 4.1 FPU/ml. Among polysaccharides (avicel, xylan, and pectin), avicel gave maximum of 6.2 FPU/ml and pretreated biomass (rice straw, wheat straw and sugarcane bagasse) produced 5.1-5.8 FPU/ml. At 7 L scale reactor, fed-batch process was designed for cellulase production using different carbon and nitrogen sources. Maximum yield of cellulases was 182 FPU/g of lactose consumed was observed in fed-batch process. The produced enzyme used for hydrolysis of acid pretreated rice straw (20% solid loading) and maximum of 60% glucan conversion was observed. RC-23-1 mutant is good candidate for large scale cellulase production and could be a model strain to study mold to yeast-like transformation.

Synergistic integration of wastewaters from second generation ethanol plant for algal biofuel production: an industrially relevant option.

The present study provides an integrated method for utilizing the wastewaters from second generation (2G) ethanol pretreatment plant for microalgal biomass and lipid production. The study was conducted using a mixture of wastewaters (referred as MW; pH 4.3) generated after washing of acidic and alkaline-soaked lignocellulosic biomass prior to pretreatment process. The growth studies indicated that the thermotolerant strain of Chlorella pyrenoidosa (C. pyrenoidosa) M18 exhibited higher cell proliferation in wastewater as compared to freshwater. About 20-25% enhancement in biomass (509 mg L-1 d-1 ± 3.09) and lipid productivity (146 mg L-1 d-1 ± 1.34) was observed in MW. The total chlorophyll content and variable fluorescence by maximum fluorescence (Fv/Fm) ratio of strain cultivated in MW were 10.32 µg mL-1 and 0.75, respectively. The use of MW also enhanced the content of saturated and monounsaturated fatty acids in total lipid. The exhausted wastewater medium obtained after harvesting the auto-flocculated biomass was also reused up to three successive growth cycles. The recycled medium without any nutrient addition could be used for two subsequent rounds with enhanced biomass (520 mg L-1 d-1 ± 4.07) and lipid (157.71 mg L-1 d-1 ± 1.09) productivities. This synergistic approach of cultivating thermotolerant microalgae with wastewater from 2G pretreatment plant provides an economical setup for development of commercial algal biofuel technology.

Enzyme systems of thermophilic anaerobic bacteria for lignocellulosic biomass conversion.

Economic production of lignocellulose degrading enzymes for biofuel industries is of considerable interest to the biotechnology community. While these enzymes are widely distributed in fungi, their industrial production from other sources, particularly by thermophilic anaerobic bacteria (growth Topt ≥ 60 °C), is an emerging field. Thermophilic anaerobic bacteria produce a large number of lignocellulolytic enzymes having unique structural features and employ different schemes for biomass degradation, which can be classified into four systems namely; 'free enzyme system', 'cell anchored enzymes', 'complex cellulosome system', and 'multifunctional multimodular enzyme system'. Such enzymes exhibit high specific activity and have a natural ability to withstand harsh bioprocessing conditions. However, achieving a higher production of these thermostable enzymes at current bioprocessing targets is challenging. In this review, the research opportunities for these distinct enzyme systems in the biofuel industry and the associated technological challenges are discussed. The current status of research findings is highlighted along with a detailed description of the categorization of the different enzyme production schemes. It is anticipated that high temperature-based bioprocessing will become an integral part of sustainable bioenergy production in the near future.

Designing a cellulolytic enzyme cocktail for the efficient and economical conversion of lignocellulosic biomass to biofuels.

Concerns about dwindling fossil fuels and their unfavorable environmental impacts shifted the global focus towards the development of biofuels from lignocellulosic feedstocks. The structure of this biomass is very complex due to which variety of enzymes (cellulolytic, hemicellulolytic, auxiliary/AA9) and proteins (e.g. swollenin) required for efficient deconstruction. Major impediments in large-scale commercial production of cellulosic ethanol are the cost of cellulases and inability of any single microorganism to produce all cellulolytic components in sufficient titers. In the recent past, various methods for reducing the enzyme cost during cellulosic ethanol production have been attempted. These include designing optimal synergistic enzyme blends/cocktail, having certain ratios of enzymes from different microbial sources, for efficient hydrolysis of pretreated biomass. However, the mechanisms underlying the development, strategies for production and evaluation of optimal cellulolytic cocktails still remain unclear. This article aims to explore the technical and economic benefits of using cellulolytic enzyme cocktail, basic enzymatic and non-enzymatic components required for its development and various strategies employed for efficient cellulolytic cocktail preparation. Consideration was also given to the ways of evaluation of commercially available and in-house developed cocktails. Discussion about commercially available cellulolytic cocktails, current challenges and possible avenues in the development of cellulolytic cocktails included.

A screening approach for assessing lytic polysaccharide monooxygenase activity in fungal strains.

BACKGROUND: Efforts to develop efficient lignocellulose-degrading enzymatic preparations have led to the relatively recent discovery of a new class of novel cellulase boosters, termed lytic polysaccharide monoxygenases (LPMOs). These enzymes are copper-dependent metalloenzymes that initiate the biomass deconstruction process and subsequently work together with cellulases, hemicellulases, and other accessory enzymes to enhance their hydrolytic action. Given their wide distribution and diversity, screening and isolation of potent LPMOs from natural fungal diversity may provide an important avenue for increasing the efficiency of cellulases and thereby decreasing cellulosic ethanol production costs. However, methods for quick screening and detection are still not widely available. In this article, a simple and sensitive method is described by combining nonhydrolytic activity enhancement followed by LC-MS-based quantitation of LPMOs. RESULTS: In this study, a screening approach has been developed for the detection of nonhydrolytic cellulase-enhancing enzymes in crude fungal supernatants. With the application of a saturating benchmark cocktail of Celluclast 1.5L, fungal isolates were selected which had the capability of hydrolyzing pretreated rice straw by their synergistic enzyme fractions. Subsequently, these fungal extracts along with an LPMO-enriched commercial enzyme were investigated for their ability to produce Type I LPMO activity. An LC-MS-based methodology was applied to quantitate gluconic acid in enzymatic hydrolysates as an indirect measurement of Type I LPMO activity. CONCLUSION: The present study describes an LC-MS-based separation method to detect and quantitate LPMO activity in a commercial enzyme. This method was also applied to screen fungal extracts. The developed screening strategy has enabled detection of LPMO activity in two industrially important Penicillium strains.

Enhanced cellulosic ethanol production via consolidated bioprocessing by Clostridium thermocellum ATCC 31924☆.

The production of bioethanol was studied by the cultivation of Clostridium thermocellum ATCC 31924 in MTC medium including crystalline cellulose as the sole substrate. The effects of key operational parameters that affect bioethanol production from microcrystalline cellulose were optimized. Under optimum conditions (pH 8.0, temperature 55 °C, inoculum size 4% (v/v) and 0.5% (w/v) substrate concentration), a maximum ethanol yield of 0.30 g ethanol/g cellulose consumed and 95.32% cellulose conversion was obtained. An inclusion of modest acetate concentration in the medium showed that carbon flux shifted away from lactate accompanied by 20% increase in ethanol production. It suggests that strain ATCC 31924 differed in its cellulose conversion efficacy and optimum pH requirements compared to the other reported strains of Clostridium thermocellum. The purified cellulosome of strain ATCC 31924 found to be rich in both cellulase and xylanase enzymes emphasizing the importance of this strain for the degradation of lignocellulosic biomass.

Bioethanol production by a xylan fermenting thermophilic isolate Clostridium strain DBT-IOC-DC21.

To overcome the challenges associated with combined bioprocessing of lignocellulosic biomass to biofuel, finding good organisms is essential. An ethanol producing bacteria DBT-IOC-DC21 was isolated from a compost site via preliminary enrichment culture on a pure hemicellulosic substrate and identified as a Clostridium strain by 16S rRNA analysis. This strain presented broad substrate spectrum with ethanol, acetate, lactate, and hydrogen as the primary metabolic end products. The optimum conditions for ethanol production were found to be an initial pH of 7.0, a temperature of 70 °C and an L-G ratio of 0.67. Strain presented preferential hemicellulose fermentation when compared to various substrates and maximum ethanol concentration of 26.61 mM and 43.63 mM was produced from xylan and xylose, respectively. During the fermentation of varying concentration of xylan, a substantial amount of ethanol ranging from 25.27 mM to 67.29 mM was produced. An increased ethanol concentration of 40.22 mM was produced from a mixture of cellulose and xylan, with a significant effect observed on metabolic flux distribution. The optimum conditions were used to produce ethanol from 28 g L-1 rice straw biomass (RSB) (equivalent to 5.7 g L-1 of the xylose equivalents) in which 19.48 mM ethanol production was achieved. Thus, Clostridium strain DBT-IOC-DC21 has the potential to perform direct microbial conversion of untreated RSB to ethanol at a yield comparative to xylan fermentation.

Cellulosic ethanol production via consolidated bioprocessing by a novel thermophilic anaerobic bacterium isolated from a Himalayan hot spring.

BACKGROUND: Cellulose-degrading thermophilic anaerobic bacterium as a suitable host for consolidated bioprocessing (CBP) has been proposed as an economically suited platform for the production of second-generation biofuels. To recognize the overall objective of CBP, fermentation using co-culture of different cellulolytic and sugar-fermenting thermophilic anaerobic bacteria has been widely studied as an approach to achieving improved ethanol production. We assessed monoculture and co-culture fermentation of novel thermophilic anaerobic bacterium for ethanol production from real substrates under controlled conditions. RESULTS: In this study, Clostridium sp. DBT-IOC-C19, a cellulose-degrading thermophilic anaerobic bacterium, was isolated from the cellulolytic enrichment cultures obtained from a Himalayan hot spring. Strain DBT-IOC-C19 exhibited a broad substrate spectrum and presented single-step conversion of various cellulosic and hemicellulosic substrates to ethanol, acetate, and lactate with ethanol being the major fermentation product. Additionally, the effect of varying cellulose concentrations on the fermentation performance of the strain was studied, indicating a maximum cellulose utilization ability of 10 g L-1 cellulose. Avicel degradation kinetics of the strain DBT-IOC-C19 displayed 94.6% degradation at 5 g L-1 and 82.74% degradation at 10 g L-1 avicel concentration within 96 h of fermentation. In a comparative study with Clostridium thermocellum DSM 1313, the ethanol and total product concentrations were higher by the newly isolated strain on pretreated rice straw at an equivalent substrate loading. Three different co-culture combinations were used on various substrates that presented two-fold yield improvement than the monoculture during batch fermentation. CONCLUSIONS: This study demonstrated the direct fermentation ability of the novel thermophilic anaerobic bacteria on various cellulosic and hemicellulosic substrates into ethanol without the aid of any exogenous enzymes, representing CBP-based fermentation approach. Here, the broad substrate utilization spectrum of isolated cellulolytic thermophilic anaerobic bacterium was shown to be of potential utility. We demonstrated that the co-culture strategy involving novel strains is efficient in improving ethanol production from real substrate.

Enhanced lipid production in thermo-tolerant mutants of Chlorella pyrenoidosa NCIM 2738.

The present study aimed to develop thermo-tolerant mutants of Chlorella pyrenoidosa NCIM 2738 for high lipids production. For this, ethyl methane sulfonate was used, which generated two effective thermo-tolerant mutants, M18 and M24 of Chlorella pyrenoidosa NCIM 2738, capable of surviving at temperature up to 47°C and showing improved lipid and biomass yields. They showed 59.62% and 50.75% increase, respectively in lipid content compared to wild type at 30°C, which could not grow at temperature above 35°C. The novelty of this study lied in incorporation of PAM Flurometry with mutagenesis to generate thermo-tolerant mutants of C. pyrenoidosa and investigating the reasons for increased yields of mutants at cellular and photosynthetic levels with the aim to use them for commercial biodiesel production.

Kinetic modeling of growth and lipid body induction in Chlorella pyrenoidosa under heterotrophic conditions.

The aim of the present work was to develop a mathematical model to describe the biomass and (total) lipid productivity of Chlorella pyrenoidosa NCIM 2738 under heterotrophic conditions. Biomass growth rate was predicted by Droop's cell quota model, while changes observed in cell quota (utilization) under carbon excess conditions were used for the modeling and predicting the lipid accumulation rate. The model was simulated under non-limiting (excess) carbon and limiting nitrate concentration and validated with experimental data for the culture grown in batch (flask) mode under different nitrate concentrations. The present model incorporated two modes (growth and stressed) for the prediction of endogenous lipid synthesis/induction and aimed to predict the effect and response of the microalgae under nutrient starvation (stressed) conditions. MATLAB and Genetic Algorithm were employed for the prediction and validation of the model parameters.

Exploring omega-3 fatty acids, enzymes and biodiesel producing thraustochytrids from Australian and Indian marine biodiversity.

The marine environment harbours a vast diversity of microorganisms, many of which are unique, and have potential to produce commercially useful materials. Therefore, marine biodiversity from Australian and Indian habitat has been explored to produce novel bioactives, and enzymes. Among these, thraustochytrids collected from Indian habitats were shown to be rich in saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs), together constituting 51-76% of total fatty acids (TFA). Indian and Australian thraustochytrids occupy separate positions in the dendrogram, showing significant differences exist in the fatty acid profiles in these two sets of thraustochytrid strains. In general, Australian strains had a higher docosahexaenoic acid (DHA) content than Indian strains with DHA at 17-31% of TFA. A range of enzyme activities were observed in the strains, with Australian strains showing overall higher levels of enzyme activity, with the exception of one Indian strain (DBTIOC-1). Comparative analysis of the fatty acid profile of 34 strains revealed that Indian thraustochytrids are more suitable for biodiesel production since these strains have higher fatty acids content for biodiesel (FAB, 76%) production than Australian thraustochytrids, while the Australian strains are more suitable for omega-3 (40%) production.

Untreated wheat straw: potential source for diverse cellulolytic enzyme secretion by Penicillium janthinellum EMS-UV-8 mutant.

Study describes the production of cellulases by Penicillium janthinellum EMS-UV-8 using untreated wheat straw (WS), treated WS (acid, alkali, steam exploded, organo-solv) and pure cellulosic substrates (avicel, cellulose-II and carboxymethyl cellulose). Severely pretreated WS and cellulose-II produced more cellulolytic enzymes than untreated samples. XRD and FTIR analysis revels that the increase in the amorphous structure of pretreated WS/cellulose increases enzyme production. Enzyme samples prepared using different substrates were used for the hydrolysis of dilute acid treated wheat straw (DATWS), steam exploded wheat straw (SEWS) and avicel. The enzyme prepared using untreated WS gave more hydrolysis of DATWS and SEWS than the enzyme prepared using pretreated WS or pure cellulosic substrates. This revels that more diverse/potential enzymes were secreted by P. janthinellum EMS-UV-8 mutant using untreated WS. This study may contribute in production of efficient enzyme mixture/cocktail by single fungal strain for economic conversion of biomass to sugars.

Enhanced cellulase production by Penicillium oxalicum for bio-ethanol application.

Present study was focused on cellulase production from an indigenously isolated filamentous fungal strain, identified as Penicillium oxalicum. Initially, cellulase production under submerged fermentation in shake flasks resulted in cellulase activity of 0.7 FPU/mL. Optimization of process parameters enhanced cellulase production by 1.7-fold and resulted in maximum cellulase activity of 1.2 FPU/mL in 8 days. Cellulase production was successfully scaled-up to 7 L fermenter under controlled conditions and incubation time was reduced from 8 days to 4 days for achieving similar cellulase titer. Optimum pH and temperature for activity of the crude enzyme were pH 5 and 50 °C, respectively. At 50 °C the produced cellulase retained approximately 50% and 26% of its activity at 48 h and 72 h, respectively. Hydrolytic efficiency of P. oxalicum was comparable to commercial cellulase preparations which indicate its great potential for application in the lignocellulose hydrolysis.

Biohydrogen production from a novel alkalophilic isolate Clostridium sp. IODB-O3.

Hydrogen producing bacteria IODB-O3 was isolated from sludge and identified as Clostridium sp. by 16S rDNA gene analysis. In this study, biohydrogen production process was developed using low-cost agro-waste. Maximum H2 was produced at 37°C and pH 8.5. Maximum H2 yield was obtained 2.54±0.2mol-H2/mol-reducing sugar from wheat straw pre-hydrolysate (WSPH) and 2.61±0.1mol-H2/mol-reducing sugar from pre-treated wheat straw enzymatic-hydrolysate (WSEH). The cumulative H2 production (ml/L), 3680±105 and 3270±100, H2 production rate (ml/L/h), 153±5 and 136±5, and specific H2 production (ml/g/h), 511±5 and 681±10 with WSPH and WSEH were obtained, respectively. Biomass pre-treatment via steam-explosion generates ample amount of WSPH which remains unutilized for bioethanol production due to non-availability of efficient C5-fermenting microorganisms. This study shows that Clostridium sp. IODB-O3 is capable of utilizing WSPH efficiently for biohydrogen production. This would lead to reduced economic constrain on the overall cellulosic ethanol process and also establish a sustainable biohydrogen production process.

Clinical application of a silk fibroin protein biologic scaffold for abdominal wall fascial reinforcement.

BACKGROUND: Preclinical studies have demonstrated that macroporous silk fibroin protein scaffolds are capable of promoting physiologically durable supportive tissue, which favors application of these engineered tissues for clinical implantation. The safety and effectiveness of a long-lasting, transitory, 510(k)-cleared purified silk fibroin biologic scaffold (SBS) are investigated for soft-tissue support and repair of the abdominal wall. METHODS: We conducted a multicenter retrospective review of all consecutive patients who underwent abdominal wall soft-tissue reinforcement with an SBS device between 2011 and 2013. Indications, comorbid conditions, surgical technique, complications, and outcomes were evaluated. RESULTS: We reviewed the records of 172 consecutive patients who received an SBS for soft-tissue support. Of those, 77 patients underwent abdominal wall fascial repair, with a mean follow-up of 18.4 ± 7.5 months. Procedures using an SBS included reinforcement of an abdominal-based flap donor site (31.2%), ventral hernia repair (53.2%), and abdominoplasty (15.6%). The overall complication rate was 6.5%, consisting of 2 wound dehiscences, 1 with device exposure, 1 seroma, 1 infection with explantation, and a perioperative bulge requiring reoperation. There were no reports of hernia. CONCLUSIONS: Postoperative complication rates after 18 months were low, and most surgical complications were managed nonoperatively on an outpatient basis without mesh removal. To our knowledge, this is the only series to report on a long-lasting, transitory SBS for abdominal wall repair and reinforcement. Procedure-specific outcome studies are warranted to delineate optimal patient selection and define potential device characteristic advantages.

Bioethanol production from wheat straw via enzymatic route employing Penicillium janthinellum cellulases.

This study concerns in-house development of cellulases from a mutant Penicillium janthinellum EMS-UV-8 and its application in separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes for bioethanol production from pre-treated wheat straw. In a 5L fermentor, the above strain could produce cellulases having activity of 3.1 FPU/mL and a specific activity of 0.83 FPU/mg of protein. In-house developed cellulase worked more efficiently in case of SSF as ethanol concentration of 21.6g/L and yield of 54.4% were obtained which were higher in comparison to SHF (ethanol concentration 12 g/L and 30.2% yield). This enzyme preparation when compared with commercial cellulase for hydrolysis of pre-treated wheat straw was found competitive. This study demonstrates that P. janthinellum EMS-UV-8 is a potential fungus for future large-scale production of cellulases.

Characterization of dielectrophoresis-aligned nanofibrous silk fibroin-chitosan scaffold and its interactions with endothelial cells for tissue engineering applications.

Aligned three-dimensional nanofibrous silk fibroin-chitosan (eSFCS) scaffolds were fabricated using dielectrophoresis (DEP) by investigating the effects of alternating current frequency, the presence of ions, the SF:CS ratio and the post-DEP freezing temperature. Scaffolds were characterized with polarized light microscopy to analyze SF polymer chain alignment, atomic force microscopy (AFM) to measure the apparent elastic modulus, and scanning electron microscopy and AFM to analyze scaffold topography. The interaction of human umbilical vein endothelial cells (HUVECs) with eSFCS scaffolds was assessed using immunostaining to assess cell patterning and AFM to measure the apparent elastic modulus of the cells. The eSFCS (50:50) samples prepared at 10MHz with NaCl had the highest percentage of aligned area as compared to other conditions. As DEP frequency increased from 100kHz to 10MHz, fibril sizes decreased significantly. eSFCS (50:50) scaffolds fabricated at 10MHz in the presence of 5mM NaCl had a fibril size of 77.96±4.69nm and an apparent elastic modulus of 39.9±22.4kPa. HUVECs on eSFCS scaffolds formed aligned and branched capillary-like vascular structures. The elastic modulus of HUVEC cultured on eSFCS was 6.36±2.37kPa. DEP is a potential tool for fabrication of SFCS scaffolds with aligned nanofibrous structures that can guide vasculature in tissue engineering and repair.

Decellularized tracheal matrix scaffold for tracheal tissue engineering: in vivo host response.

BACKGROUND: The authors have previously demonstrated promising results with tissue engineered trachea in vitro using decellularized matrix scaffolds. The present study aims to investigate the applicability of the construct in vivo. METHODS: Tracheae harvested from Brown Norway rats (donor) and Lewis rats (recipient) were decellularized with repeated detergent-enzymatic treatment cycles. Decellularized Brown Norway tracheal matrix scaffolds were seeded with Lewis rat stem cell-derived chondrocytes externally and tracheal epithelial cells internally to generate a bilaminated tracheal construct. Brown Norway tracheal matrix scaffolds (n = 6), Lewis rat scaffolds (n = 6), and the engineered constructs (n = 3) were implanted subcutaneously in Lewis rats and observed for 4 weeks. Fresh Brown Norway (n = 6) and Lewis rat (n = 6) tracheae were implanted as controls. Histologic analysis for macrophage, CD8, and CD4 cell infiltration was performed. RESULTS: Allogeneic decellularized matrix scaffold showed significantly decreased macrophage, CD8+ and CD4+ cell infiltration compared with tracheal allografts, and demonstrated similar level of cell infiltration to syngeneic decellularized matrix scaffold. No significant differences in macrophage infiltration were observed between syngeneic decellularized matrix scaffolds and tracheal isografts. The engineered constructs achieved complete epithelial cell coverage and preserved lumen patency; however, chondrocytes failed to repopulate the cartilaginous matrix with statically seeding stem cell on scaffold. CONCLUSIONS: Decellularized tracheal matrix scaffold did not induce significant allograft rejection or foreign body reaction in vivo. Although the construct supported reepithelialization, stem cell-derived chondrocytes failed to engraft in the heterotopic environment and represent a focus of future investigations.