Type of endothelial cells affects HepaRG cell acetaminophen metabolism in both 2D and 3D porous scaffold cultures.

Recent advances in developing in vitro tissue models show that function of hepatocytes is altered in when cultured in 3D configuration and co-culturing with various non-parenchymal cells. However, tissue source for such non-parenchymal cells on viability and metabolic products of hepatocytes have not been explored. In this study, we evaluated the effect of 2D and 3D cultures either with HepaRG cells alone or in combination with liver sinusoidal endothelial cells (LSECs) and human umbilical vein ECs (HUVECs). For 3D cultures, we used chitosan-gelatin porous structures formed by freeze-drying. We cultured cells for 8 days before challenging with 1 mm acetaminophen (APAP) and assessed APAP, APAP-sulfate and APAP-glucuronide for 24 hours at 6-hour time intervals using high-performance liquid chromatography. We used multiple methods (phase contrast, confocal and scanning electron microscopy and histology via hematoxylin and eosin staining) to ensure cell distribution. We also measured total protein content and albumin secretion and viability. HUVEC 3D co-cultures showed the lowest HepaRG cell viability, while both 2D and 3D LSEC co-cultures had highest HepaRG cell viability. In addition, 3D cultures had significantly higher EC viability relative to 2D cultures. Further, HUVEC co-cultures showed reduced total protein content and albumin expression as early as day 4. However, urea production on a total protein content basis did not change. In addition, LSEC 3D co-cultures had the highest APAP conversion with reduced APAP-sulfate and APAP-glucuronide formation. CYP3A4 was higher in co-culture with HUVEC for 2D and 3D cultures. In conclusion, HepaRG cells with EC co-cultures demonstrated sensitivity to the EC line used.

Multiple approaches to predicting oxygen and glucose consumptions by HepG2 cells on porous scaffolds in an axial-flow bioreactor.

In this study, the distribution of oxygen and glucose was evaluated along with consumption by hepatocytes using three different approaches. The methods include (i) Computational Fluid Dynamics (CFD) simulation, (ii) residence time distribution (RTD) analysis using a step-input coupled with segregation model or dispersion model, and (iii) experimentally determined consumption by HepG2 cells in an open-loop. Chitosan-gelatin (CG) scaffolds prepared by freeze-drying and polycaprolactone (PCL) scaffolds prepared by salt leaching technique were utilized for RTD analyses. The scaffold characteristics were used in CFD simulations i.e. Brinkman's equation for flow through porous medium, structural mechanics for fluid induced scaffold deformation, and advection-diffusion equation coupled with Michaelis-Menten rate equations for nutrient consumption. With the assumption that each hepatocyte behaves like a micro-batch reactor within the scaffold, segregation model was combined with RTD to determine exit concentration. A flow rate of 1 mL/min was used in the bioreactor seeded with 0.6 × 10(6) HepG2 cells/cm(3) on CG scaffolds and oxygen consumption was measured using two flow-through electrodes located at the inlet and outlet. Glucose in the spent growth medium was also analyzed. RTD results showed distribution of nutrients to depend on the surface characteristics of scaffolds. Comparisons of outlet oxygen concentrations between the simulation results, and experimental results showed good agreement with the dispersion model. Outlet oxygen concentrations from segregation model predictions were lower. Doubling the cell density showed a need for increasing the flow rate in CFD simulations. This integrated approach provide a useful strategy in designing bioreactors and monitoring tissue regeneration.

Increased matrix synthesis by fibroblasts with decreased proliferation on synthetic chitosan-gelatin porous structures.

Influence of mechanical characteristics and matrix architecture of substrates used in cell culture is an important issue to tissue engineering. Chitosan-based materials have been processed into porous structures, injectable gels and membranes, and are investigated to regenerate various tissues. However, the effect of these structures on cell growth and matrix production in accordance with each of the differing scaffolds has not been examined. We investigated the influence of porous structures, hydrogels, and membranes on the growth of normal human fibroblasts and their matrix production in a serum-free system. We used chitosan alone and in combination with gelatin. Injectable hydrogels were prepared using 2-glycerol phosphate. From the same solution, porous scaffolds and membranes were formed using controlled rate freezing and lyophilization, and air-drying, respectively. Fibroblast growth was evaluated on the 4th and 10th days using flow cytometry and CFDA-SE pre-staining. Cell morphology was assessed using actin and nucleus staining. Total protein content, collagen, tropoelastin, and MMP2/MMP-9 activity in the media supernatant were assessed by BCA, Sircol™, Fastin Elastin, and fluorogeneic peptide assays. Collagen accumulated in the matrix was assessed by Sircol™ assay after pepsin/acetic acid digestion and by Masson's Trichrome staining. These results showed increased viability of fibroblasts on chitosan-gelatin porous scaffold with decreased proliferation relative to tissue culture plastic (TCP) surface despite the cells showing spindle shape. The total protein, collagen, and tropoelastin contents were higher in the spent media from chitosan-gelatin porous scaffolds compared to other conditions. MMP2/MMP9 activity was comparable to TCP. An increase in collagen content was also observed in the matrix, suggesting increased matrix deposition. In summary, matrix production is influenced by the form of chitosan structures, which significantly affects the regenerative process.

Recent advances in the combination delivery of drug for leukemia and other cancers.

Introduction: Combination therapy has been explored for its potential to reduce or eliminate multidrug resistance in treating different types of cancer including leukemia. Nutraceutical, small molecular drugs, and small interfering ribonucleic acid (siRNA) are some of the effective drugs. In order to avoid off-site targeting, reduce the dosage required, and increase the half-life of the drug in the circulation system, drug delivery vehicles, such as nanoparticles and microfibers have been explored.Areas covered: This review summarizes various therapies utilized in treating leukemia based on their effectiveness in inducing protein inhibition and/or apoptosis. In particular, treatment effectiveness using combination therapy using various devices is addressed. Recently explored drug delivery methods are reviewed, providing examples and their applications in cancer treatment. The drug listing, delivery systems classifications, along with the general modeling approach in this review, provide, to a full extent, a basis for cancer drug delivery future studies.Expert opinion: The reviewer's opinion tackles the potential of using a multi-delivery system to deliver multiple drugs, providing better control upon drug release and targeting. Both local and systemic delivery are considered and explored for their potential targets. Researchers are advised to pre-consider all aspects associated with their desired delivery method.

Modeling the impact of fluid flow on resveratrol release from electrospun fibers.

Using electrospun fibers to deliver therapeutic agents has gained significant attention in various applications including cancer treatment and tissue regeneration. However, the effect of fluid flow and uptake by cells on the concentration profile is not well understood. In this study, we evaluated the release of lipophilic resveratrol from poly(ε-caprolactone) (PCL)-gelatin (GT) electrospun fibers experimentally and by using computational fluid dynamics (CFD). Resveratrol containing PCL-GT electrospun fibers were formed and used in a custom-built tubular bioreactor, to assess flow effect on concentration profile over 5 days. CFD model was developed to simulate release in both static cultures and under fluid flow conditions. Resveratrol stability in the culture medium and uptake by human umbilical vein endothelial cells and K562 cells over 3 days were used in the model. The concentration profile as a function of time was simulated and validated by experiments. The effects of inlet velocity, cellular uptake rate, bioreactor's length, and surrounding tissue porosity were assessed. The release profile was mainly affected by cellular uptake and the presence of porous media. The model suggests that the perfusion velocity might not have a significant effect relative to the cellular uptake rate and porosity of the surrounding tissue.

Modeling nutrient consumptions in large flow-through bioreactors for tissue engineering.

Flow-through bioreactors are utilized in tissue regeneration to ensure complete nutrient distribution and apply defined hydrodynamic stresses. The fundamental concepts in designing these bioreactors for regenerating large high aspect ratio tissues (large surface area relative to the thickness of the matrix such as skin, bladder, and cartilage) are not well defined. Further, tissue regeneration is a dynamic process where the porous characteristics change due to proliferation of cells, de novo deposition of matrix components, and degradation of the porous architecture. These changes affect the transport characteristics and there is an imminent need to understand the influence of these factors. Using computational fluid dynamic tools, changes in the pressure drop, shear stress distribution and nutrient consumption patterns during tissue regeneration were assessed in rectangular and circular reactors described by Lawrence et al. [Biotechnol Bioeng 2009;102(3):935-947]. Further, six new designs with different inlet and outlet shapes were analyzed. The fluid flow was defined by the Brinkman equation on the porous regions using the pore characteristics of 85 microm and 120 pores/mm(2). The minimum flow requirements to satisfy nutrient (oxygen and glucose) requirements for three different cell types (SMCs, chondrocytes, and hepatocytes) was evaluated using convective diffusion equation. For consumption reaction, the Michaelis-Menten rate law was used, with constants (k(m) and v(m) values) extracted from literature. Simulations were performed by varying the flow rate as well as the cell number. One of the circular reactors with semicircular inlet and outlet shape decreased (i) non-uniformity in hydrodynamic stress within the porous structure and (ii) non-uniform nutrient distribution. All cell types showed increased consumption of oxygen than glucose. Hepatocytes needed a very high flow rate relative to other cell types. Increase in cell number suggested a need for increasing the flow in circular reactors.

Flow dynamics in bioreactors containing tissue engineering scaffolds.

Bioreactors are widely used in tissue engineering as a way to distribute nutrients within porous materials and provide physical stimulus required by many tissues. However, the fluid dynamics within the large porous structure are not well understood. In this study, we explored the effect of reactor geometry by using rectangular and circular reactors with three different inlet and outlet patterns. Geometries were simulated with and without the porous structure using the computational fluid dynamics software Comsol Multiphysics 3.4 and/or ANSYS CFX 11 respectively. Residence time distribution analysis using a step change of a tracer within the reactor revealed non-ideal fluid distribution characteristics within the reactors. The Brinkman equation was used to model the permeability characteristics with in the chitosan porous structure. Pore size was varied from 10 to 200 microm and the number of pores per unit area was varied from 15 to 1,500 pores/mm(2). Effect of cellular growth and tissue remodeling on flow distribution was also assessed by changing the pore size (85-10 microm) while keeping the number of pores per unit area constant. These results showed significant increase in pressure with reduction in pore size, which could limit the fluid flow and nutrient transport. However, measured pressure drop was marginally higher than the simulation results. Maximum shear stress was similar in both reactors and ranged approximately 0.2-0.3 dynes/cm(2). The simulations were validated experimentally using both a rectangular and circular bioreactor, constructed in-house. Porous structures for the experiments were formed using 0.5% chitosan solution freeze-dried at -80 degrees C, and the pressure drop across the reactor was monitored.

Targeted cancer treatment using a combination of siRNA-liposomes and resveratrol-electrospun fibers in co-cultures.

In this study, we evaluated a novel combination of drug delivery devices composed of holo-transferrin conjugated liposomes for siRNA (36 nM) delivery, and electrospun polycaprolactone (PCL)-gelatin (GT) microfibers for resveratrol (40 µM) release. Single- and co-cultures of cancerous K562 cells and human umbilical vein endothelial cells (HUVECs) were used to test the efficacy and targeting over eight days. BCR-ABL siRNA-encapsulated (36 nM) holo-transferrin-conjugated PEG-liposomes were characterized using dynamic light scattering, and transmission electron microscopy. RT-qPCR was performed to assess the silencing BCR-ABL gene. Two treatment protocols were explored: i) simultaneous administration ii) delayed liposomes addition by three days based on resveratrol release profile. Formed liposomes were 123 (±6.65) nm in diameter, holo-transferrin conjugation efficiency was 85.9 (±7.30)%, and siRNA loading efficiency was 92.3 (±2.57)%. Sphingosine-1-phosphate (S1P) content was analyzed by ELISA. Targeted siRNA release in combination with resveratrol release was more potent and has long-term effects compared to bolus doses. Delayed addition of liposomes increased non-viability of K562 cells to 92.7 (±2.00)% and 94.32 (±1.70)%, in the absence and presence of HUVECs, respectively. HUVECs non-viability level was significantly lower. Using two different delivery devices approach has a broader impact on cancer treatment.

Formation of Stem Cell Aggregates and Their Differentiation on Surface-Patterned Hydrogels Based on Poly(2-hydroxyethyl Methacrylate).

Micropillar patterns were fabricated and used to study cell adhesion, morphology, and function. Micropillars were produced in poly(2-hydroxyethyl methacrylate (HEMA)/N,N-(dimethylaminoethyl)methacrylate (DMAEMA)/tetraethylene glycol dimethacrylate (TEGDMA)) hydrogels using soft lithography, had dimensions of 1 µm diameter, and were either 2.05 or 4.91 µm tall. The patterned hydrogel substrates increased adhesion and induced the formation of cellular aggregates. Digital micrographs were used to quantify aggregate size and number. Differentiation of hMSCs toward adipocytes and chondrocytes was performed using the respective complete culture and differentiation medium for 2 weeks. Cells were stained for Oil red O, Alcian blue, and Type II collagen. Hydrogel substrates supported the differentiation of hMSCs to adipocytes and chondrocytes. The taller micropillar patterns supported the attachment and growth of larger aggregates and were more amenable to aid chondrogenic differentiation.

Immunomodulatory response of layered small intestinal submucosa in a rat bladder regeneration model.

Based on the hypothesis that bioscaffold permeability is a major factor in determining the outcome of histologically complete and functional bladder regeneration, we evaluated regeneration processes of four-layer porcine small intestinal submucosa (SIS) construct; and compared results between rat bladders augmented with single-layer SIS bioscaffolds. Sprague-Dawley female rats were subjected to hemi-cystectomy followed by anastomosis of a patch of either single- or four-layer porcine SIS. Permeability was analyzed in situ using magnetic resonance imaging (MRI) at post-operative days 7 and 14. Bladder sections excised at days 7, 14, 28, and 56 post-operation Samples were assessed by H&E and Masson's trichrome stains. Urothelial differentiation was analyzed using cytokeratin AE1/AE3, and uroplakin III (UPIII). In addition, quantitative and qualitative evaluations of neutrophils, mast cells, eosinophils, and macrophages were performed using anti-myeloperoxidase, Alcian blue, Giemsa stain, and anti-CD68 staining methods, respectively. Four-layer SIS was consistently impermeable as evidenced by the absence of intravesical administered gadolinium with diethylenetriaminepentacetate (Gd-DTPA) contrast signal in peripheral regions of augmented bladders compared with single-layer SIS bioscaffold. Elevated and sustained eosinophil and neutrophil infiltrations were prominent in four-layered SIS-augmented bladders compared with single-layer SIS with comparable impermeability. Delayed but consistent urothelial regeneration and differentiation were observed in four-layer SIS-augmented bladders; and urothelial differentiation was observed at day 56 post-augmentation. In conclusion, four-layer SIS enacts an elevated inflammatory response along with extended urothelial regeneration. Four-layer SIS may activate a different but yet to be identified mechanism for inflammatory responses. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1960-1969, 2019.

Influence of controlled release of resveratrol from electrospun fibers in combination with siRNA on leukemia cells.

In this study, we evaluated the possibility of i) local release of resveratrol from poly(ε-caprolactone) (PCL) and gelatin (GT) electrospun fibers and ii) combining (i) with siRNA designed to downregulate BCR-ABL pathway on K562 cancer cells. Initially, K562 cell culture experiments were performed using various bolus doses of resveratrol in combination with siRNA for 3 days using a factorial design of experiments approach. Resveratrol content was analyzed using HPLC and cell viability was assessed using Annexin V (Non-viable), and Propidium Iodide (PI) (Necrotic) based flow cytometry. Coaxial electrospun fibers with resveratrol were made using 1:1 PCL-GT blends in different configurations: single fibers and coaxial fibers with same polymer blends, or with PCL inner core. Loading efficiency and release profile over five days were analyzed. Based on release profile, K562 cell viability with fibers was analyzed over eight days. Dose dependent cell death was observed with bolus resveratrol and siRNA in the culture. However, resveratrol content depleted significantly when added directly to solution. The combination therapy was additive in solution. SEM analysis showed no phase separation of components and resveratrol loading efficiency varied from 77% to 88% in different configurations; 95% of resveratrol was released by day five. Permeability of resveratrol showed no significant dependency on fiber configuration. After 8 days, non-viable cell percentages with controlled release were similar to that at three-day bolus dose of resveratrol. However, siRNA interacted with the fibers, resulting in reduced effect on cells. Loading resveratrol into electrospun fibers provides a localized delivery at therapeutic level, and increased resveratrol's apoptotic effect. Using single fibers is sufficient for controlled release.

Investigation of the Roles of Plasma Species Generated by Surface Dielectric Barrier Discharge.

As an emerging sterilization technology, cold atmospheric plasma offers a dry, non-thermal, rapid process that is minimally damaging to a majority of substrates. However, the mechanisms by which plasma interacts with living cells are poorly understood and the plasma generation apparatuses are complex and resource-intensive. In this study, the roles of reactive oxygen species (ROS), nitric oxide (NO), and charged particles (ions) produced by surface dielectric barrier discharge (SDBD) plasma on prokaryotic (Listeria monocytogenes (Gram-positive)) and eukaryotic (human umbilical vein endothelial cells (HUVEC)) cellular function were evaluated. HUVEC and bacterial oxidative stress responses, the accumulation of nitrite in aqueous media, air ion density, and bacterial inactivation at various distances from SDBD actuators were measured. SDBD actuator designs were also varied in terms of electrode number and length to evaluate the cellular effects of plasma volume and power distribution. NO and ions were found to contribute minimally to the observed cellular effects, whereas ROS were found to cause rapid bacterial inactivation, induce eukaryotic and prokaryotic oxidative stress, and result in rapid oxidation of bovine muscle tissue. The results of this study underscore the dominance of ROS as the major plasma generated species responsible for cellular effects, with ions and RNS having a secondary, complimentary role.

Reduced urothelial regeneration in rat bladders augmented with permeable porcine small intestinal submucosa assessed by magnetic resonance imaging.

Augmentation enterocystoplasty remains the gold standard surgical bladder reconstruction procedure to increase the capacity and compliance of dysfunctional bladders. Since the use of the patient's intestine has severe risks of complications, alternative biodegradable matrices have been explored. Porcine small intestinal submucosa (SIS) has gained immense interests in bladder reconstruction due to its favorable properties. However, trials have shown inconsistent regeneration with SIS, attributed to the heterogeneity in microstructures and mechanical properties. We hypothesize that uneven SIS permeability to urine is a factor responsible for the inconsistency. We measured permeability to urine in situ using a contrast enhanced-magnetic resonance imaging (MRI), and evaluated urothelium regeneration using immunohistochemical staining of urothelial cell markers in SIS-augmented rat bladders. Results showed significant differences in permeability among SIS-augmented rat bladders. Commercial SIS scaffolds were then categorized into nonleaky and leaky groups based on MRI results. Hematoxylin and eosin staining showed higher numbers of inflammatory cells in leaky SIS on day 14 relative to nonleaky SIS. In addition, trichrome staining showed major changes in the distribution of collagen on day 28 between SIS-augmented bladder groups. Furthermore, expressions of urothelium-associated markers (cytokeratins AE1/AE3, claudin 4, and uroplakin III) were completed in bladders augmented with nonleaky SIS, whereas limited urothelial differentiation was noticed in leaky SIS-augmented bladders at post-augmentative day 14. These results show that scaffold permeability to urine may be responsible for variations in regenerative capacity of porcine SIS. Applications of MRI technique will be helpful to understand a relationship between biomaterial property and regenerative capacity. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1778-1787, 2018.

Blending chitosan with polycaprolactone: porous scaffolds and toxicity.

The preparation and characterization of porous scaffolds from chitosan-PCL blends by freeze extraction, freeze gelation and freeze drying is reported. Using freeze extraction, stable structures were obtained only from PCL, but these were not porous. No stable scaffolds were obtained using the freeze gelation process. Stable scaffolds of chitosan/PCL mixtures could not be obtained using 77% acetic acid by any of these techniques. With 25% aqueous acetic acid, stable scaffolds of chitosan/PCL mixtures were obtained by the freeze drying technique. The stability and pore morphology of freeze dried scaffolds were dependent on the relative mass ratio of chitosan and PCL. A chorioallantoic membrane assay showed that formed 3D chitosan/PCL mixtures were not toxic to vasculature.

Recent advances in multiaxial electrospinning for drug delivery.

Electrospun fibers have seen an insurgence in biomedical applications due to their unique characteristics. Coaxial and triaxial electrospinning techniques have added new impetus via fabrication of multilayered nano and micro-size fibers. These techniques offer the possibility of forming fibers with features such as blending, reinforced core, porous and hollow structure. The unique fabrication process can be used to tailor the mechanical properties, biological properties and release of various factors, which can potentially be useful in various controlled drug delivery applications. Harvesting these advantages, various polymers and their combinations have been explored in a number of drug delivery and tissue regeneration applications. New advances have shown the requirement of drug-polymer compatibility in addition to drug-solvent compatibility. We summarize recent findings using both hydrophilic and hydrophobic (or lipophilic) drugs in hydrophobic or hydrophilic polymers on release behavior. We also describe the fundamental forces involved during the electrospinning process providing insight to the factors to be considered to form fibers. Also, various modeling efforts on the drug release profiles are summarized. In addition new developments in the immune response to the electrospun fibers, and advances in scale-up issues needed for industrial size manufacturing.

Modeling the permeability of multiaxial electrospun poly(ε-caprolactone)-gelatin hybrid fibers for controlled doxycycline release.

Recent advances in electrospinning allow the formation of multiple layers of micro and nanosize fibers to regulate drug/therapeutic agent release. Although there has been significant progress in fiber formation techniques and drug loading, fundamental models providing insights into controlling individual permeabilities is lacking. In this regard, we first explored forming coaxial hybrid fibers from hydrophobic poly(ε-caprolactone) (PCL) and hydrophilic gelatin (GT) in three different configurations, and the release of hydrophilic doxycycline (Dox) at 37°C over five days. Triaxial fibers were also formed with a GT layer between PCL/GT layers. Fibers were analyzed for fiber thickness, matrix porosity and thickness, surface morphologies, internal structures, stability in hydrated condition, viability and attachment of human adipocyte stem cells (hASC). Formed fibers were 10-30µm in diameter. hASC were viable, and showed attachment. Various release profiles were obtained from these fibers based on the combination of the core and shell polymers over five days. Using fiber characteristics and release profiles from each configuration, we obtained the overall permeability using Fick's first law and then individual layer permeability using resistance in series model. Calculated overall permeability showed dependency on fiber thickness and partition coefficient of the drug in the region where it was loaded. Our modeling approach helps in optimizing the electrospinning process, drug loading, and polymer solution configuration in regulating controlled release of a drug.

Bioprinted chitosan-gelatin thermosensitive hydrogels using an inexpensive 3D printer.

The primary bottleneck in bioprinting cell-laden structures with carefully controlled spatial relation is a lack of biocompatible inks and printing conditions. In this regard, we explored using thermogelling chitosan-gelatin (CG) hydrogel as a novel bioprinting ink; CG hydrogels are unique in that it undergoes a spontaneous phase change at physiological temperature, and does not need post-processing. In addition, we used a low cost (<$800) compact 3D printer, and modified with a new extruder to print using disposable syringes and hypodermic needles. We investigated (i) the effect of concentration of CG on gelation characteristics, (ii) solution preparation steps (centrifugation, mixing, and degassing) on printability and fiber formation, (iii) the print bed temperature profiles via IR imaging and grid-based assessment using thermocouples, (iv) the effect of feed rate (10-480 cm min-1), flow rate (15-60 µl min-1) and needle height (70-280 µm) on fiber size and characteristics, and (v) the distribution of neuroblastoma cells in printed fibers, and the viability after five days in culture. We used agarose gel to create uniform print surfaces to maintain a constant gap with the needle tip. These results showed that degassing the solution, and precooling the solution was necessary for obtaining continuous fibers. Fiber size decreased from 760, to 243 µm as the feed rate increased from 10 to 100 cm min-1. Bed temperature played the greatest role in fiber size, followed by feed rate. Increased needle height initially decreased fiber size but then increased showing an optimum. Cells were well distributed within the fibers and exhibited excellent viability and no contamination after 5 d. Overall we printed 3D, sterile, cell-laden structures with an inexpensive bioprinter and a novel ink, without post-processing. The bioprinter described here and the novel CG hydrogels have significant potential as an ink for bioprinitng various cell-laden structures.

Three-dimensional cell colonization in a sulfate rich environment.

Glycosaminoglycans (GAGs) have been explored for regenerating various tissues due to their involvement in diverse bioregulatory activity. However, understanding their influence on cell colonization in three-dimension (3-D) has been difficult due to variation in their molecular weight, degree of sulfation, and lack of in vitro models. This research focused on developing an in vitro model and evaluating the influence of MW (5, 10, and 500 kDa) of negatively charged dextran sulfate (DS), a semisynthetic GAG analog, on cell colonization. DS was combined with chitosan, a positively charged polymer in solution and porous 3-D matrices were formed inside 24-well plates using controlled rate freezing and lyophilization technique by two schemes: (i) chitosan structures were formed and then allowed to react with DS; (ii) DS was reacted with chitosan in solution and then matrices were formed. Scanning electron microscopy analysis showed that forming matrices after reacting DS with chitosan was more suitable for tissue regeneration. Analysis for the quantity and stability of DS by toluidine blue assay indicated significant presence of DS in the 3-D matrices even after seven days of incubation in phosphate buffered saline solution. Matrices formed by reacting 4% 5 kDa, 2% 10 kDa and 1% 500 kDa DS solution with chitosan had optimum porosity and mechanical stability. Next, 25,000 fibroblasts per matrix were seeded onto 3-D matrices and analyzed for proliferation by MTT-formazan assay, cytoskeletal organization by actin staining, and histological analysis by H/E staining. These results showed that cell growth was better on low MW containing 2-D membranes but high MW DS containing 3-D matrix supported cell growth similar to chitosan. Also, cells showed peripheral actin distribution in 3-D matrices. Analysis of fibronectin binding by ELISA showed negligible binding to all the DS-containing matrices, unlike chitosan. In summary, results show cell colonization on negatively charged matrices, similar to chitosan.

Characterization of emulsified chitosan-PLGA matrices formed using controlled-rate freezing and lyophilization technique.

This study evaluated the formation of chitosan-50:50 poly-lactic-co-glycolic acid (PLGA) blend matrices using controlled-rate freezing and lyophilization technique (CRFLT). An emulsion system was used in the presence of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), a cellular component, as a stabilizer. Blended scaffolds showed an open pore morphology and homogenous interdispersion of PLGA and chitosan. Forming emulsions after dissolving PLGA in chloroform, benzene, or methylene chloride indicated better emulsion stability with benzene and chloroform. Scaffolds formed by freezing at -20, -78, and -196 degrees C from these emulsions showed significant influence of the solvent and freezing temperature on the microarchitecture of the scaffold. By controlling the concentration of chitosan, scaffolds with greater than 90% porosity were attained. Since the two polymers degrade by different mechanisms, formed scaffolds were analyzed for degradation characteristics for 4 weeks in presence of 10 mg/L lysozyme. These results showed no significant difference in the weight loss and dimension changes, as all scaffolds showed significant (a) weight loss and (b) nearly 60% reduction in volume. Further, pH of the incubation media decreased in all the samples. When cellular activity of green fluorescence protein-transfected smooth muscle cells was analyzed, no apparent cytotoxicity was observed. However, the cell spreading area decreased. In summary, these results show promising potential in tissue engineering and drug delivery applications.

In vitro characterization of acelluar porcine adipose tissue matrix for use as a tissue regenerative scaffold.

This study evaluated a novel approach to decellularizing porcine adipose tissue while preserving its 3-D architecture. An ethanol-water mixture was used as a solvent to remove lipids and the number of freeze-thaw cycles (1-7), ethanol concentration, and tissue thickness were tested. Trypsin incubation time (1-3 h) and xylene immersion time were investigated separately. Processed sample microarchitecture was analyzed via scanning electron microscope, cellular content was analyzed via hematoxylin and eosin (H&E) staining, and DNA content was analyzed using gel electrophoresis. Tensile testing and five-stage incremental stress-relaxation testing was performed in phosphate-buffered saline at 37°C. Human neuroblasts were seeded and evaluated for infiltration and attachment over 8 days. Four cycles of freeze-thaw in 50% ethanol-water mixture removed one-third of the lipids. Microarchitecture showed the presence of pores, capillary channels, and lack of sidedness; H&E micrographs confirmed unaltered morphology and absence of cells. Incubation for 1.5 h in trypsin removed 99.5% DNA from delipidized samples. An average of 40% rehydration swelling, an elastic modulus of 324(±141) kPa, and an ultimate tensile strength of 87.4(±23.1) kPa were observed. The matrix exhibited strain hardening behavior similar to small intestinal submucosa. Cells successfully infiltrated and spread in the decellularized scaffold. Removal of lipids significantly reduced incubation in trypsin EDTA. In summary, the acellular matrix shows significant potential as a new template for tissue regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3127-3136, 2016.