Biomedical subjects

Sundararajan V Madihally

Publications and source records attributed to Sundararajan V Madihally.

10 recordsLinked to original sources

PubMed2006

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.

Animals↗

PubMed2006

Antiproteolytic action of orally delivered insulin using pH-responsive hydrogels in a rat burn model.

Subcutaneously delivered small doses of insulin has shown beneficial effects on burn injury-induced muscle wasting and wound healing. To improve the method of insulin treatment for clinical settings, this study investigated the effect of insulin delivered orally using pH-responsive poly(methacrylic-g-ethylene glycol) (P(MAA-g-EG)) hydrogels in a 20% total burn surface area rat burn injury model. P(MAA-g-EG) were synthesized in-house and insulin release characteristics were performed in vitro. Young rats weighing 80-150 g were subjected to 15-20% total body surface area burn injury and treated with insulin-containing hydrogels enclosed in gelatin capsules for 3 days. The dosage was adjusted to match 0.25 U (day 1), 0.5U (day 2), and 1.0 U (day 3) per 100 grams of body weight. All animals were housed in metabolic cages and their physical activity, body weight, food consumption, water uptake, circulating glucose levels, and urinary tyrosine content were monitored for 4 to 15 days after burn. Results show that the orally delivered insulin restored the body weight of burned rats and influenced wound healing, similar to subcutaneous delivery. Measured glucose levels showed significantly less perturbation, suggesting the possibility of increasing the dosage. In conclusion, muscle wasting can be significantly inhibited by the oral administration of insulin using pH-responsive hydrogels.

Administration, Oral↗

PubMed2006

Effect of spatial architecture on cellular colonization.

The spatial cell-material interaction remains vital issue in forming biodegradable scaffolds in Tissue Engineering. In this study, to understand the influence of spatial architecture on cellular behavior, 2D and 3D chitosan scaffolds of 50-190 kD and >310 kD MW were synthesized through air drying and controlled rate freezing/lypohilization technique, respectively. In addition, chitosan was emulsified with 19, 76, and 160 kD 50:50 poly lactide-co-glycolide (PLGA) using 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) as stabilizer. 2D and 3D scaffolds were formed by air drying and lyophilization as before. Tensile and compressive properties of films and scaffolds were analyzed in wet conditions at 37 degrees C. Alterations in the cell spreading, proliferation, and cytoskeletal organization of human umbilical vein endothelial cells (HUVECs) and mouse embryonic fibroblasts (MEFs) were studied. These results showed that the formed 3D chitosan scaffolds had interconnected open pore architecture (50-200 microm size). HUVECs and MEFs had reduced spreading areas and circular morphology on 2D chitosan membranes compared with 3D chitosan scaffolds. The fluorescence photomicrographs for actin (using Alexa Fluor 488 phalloidin) and cytoplasm staining (using carboxyfluorescein diacetate-succinimidyl ester) demonstrated that the cells spread within 3D chitosan matrix. 2D and 3D emulsified chitosan and chitosan/PLGA scaffolds reduced the spreading of HUVECs and MEFs even further. Proliferation results, analyzed via MTT-Formazan assay and BrdU uptake assay, correlated with the spreading characteristics. The reductions in cell spreading area on emulsified surfaces were not detrimental to the viability and endocytic activity but to proliferation. The observed alterations in cellular colonization are in part due to the substrate stiffness and surface topography. In summary, these results suggest a significant influence of spatial architecture on cellular colonization.

Cell Culture Techniques↗

PubMed2006

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.

Biocompatible Materials↗

PubMed2006

Blending chitosan with polycaprolactone: effects on physicochemical and antibacterial properties.

Chitosan is a well sought-after polysaccharide in biomedical applications and has been blended with various macromolecules to mitigate undesirable properties. However, the effects of blending on the unique antibacterial activity of chitosan as well as changes in fatigue and degradation properties are not well understood. The aim of this work was to evaluate the anti-bacterial properties and changes in physicochemical properties of chitosan upon blending with synthetic polyester poly(epsilon-caprolactone) (PCL). Chitosan and PCL were homogeneously dissolved in varying mass ratios in a unique 77% acetic acid in water mixture and processed into uniform membranes. When subjected to uniaxial cyclical loading in wet conditions, these membranes sustained 10 cycles of predetermined loads up to 1 MPa without break. Chitosan was anti-adhesive to Gram-positive Streptococcus mutans and Gram-negative Actinobacillus actinomycetemcomitans bacteria. Presence of PCL compromised the antibacterial property of chitosan. Four-week degradation studies in PBS/lysozyme at 37 degrees C showed initial weight loss due to chitosan after which no significant changes were observed. Molecular interactions between chitosan and PCL were investigated using Fourier transform infrared spectroscopy (FTIR) which showed no chemical bond formations in the prepared blends. Investigation by wide-angle X-ray diffraction (WAXD) indicated that the crystal structure of individual polymers was unchanged in the blends. Dynamic mechanical and thermal analysis (DMTA) indicated that the crystallinity of PCL was suppressed and its storage modulus increased with the addition of chitosan. Analysis of surface topography by atomic force microscopy (AFM) showed a significant increase in roughness of all blends relative to chitosan. Observed differences in biological and anti-bacterial properties of blends could be primarily attributed to surface topographical changes.

Anti-Bacterial Agents↗

PubMed2005

Physical characteristics of small intestinal submucosa scaffolds are location-dependent.

Using biodegradable scaffolds as an alternative to engineer new tissues has become an attractive candidate in various transplantation protocols. In particular, small intestinal submucosa (SIS), a dense connective matrix harvested from the small intestine, has gained attention due to a number of favorable properties. However, use of SIS is constrained by obtaining reliable, reproducible products in large-scale preparations that affect the regenerative process. To better understand the heterogeneous nature of SIS, this study focused on evaluating the location-dependent alterations in the physical characteristics of the matrices harvested from distal and proximal ends and processed in-house (referred as hand-made). Additionally, results were compared with a commercially available machine-made Cook SIS. Tensile properties during monotonic loading and cyclical loading were compared in wet conditions. Furthermore, permeability of these membranes to urea was analyzed using a custom-built chamber, and the microarchitecture was analyzed via scanning electron microscopy. These results showed that distal samples were more elastic and less permeable to urea relative to other samples. However, permeability in each sample was direction-dependent, that is, mucosal to serosal direction was less permeable compared to sorasal to mucosal direction in all the samples. Cook SIS was more susceptible to cyclical loading and had a shorter range of load carrying capacity. In summary, results show that physical characteristics of SIS are location-dependent.

Absorbable Implants↗

PubMed2005

Characterization of chitosan-polycaprolactone blends for tissue engineering applications.

The objective of this work was to study the effect of blending chitosan with poly(epsilon-caprolactone) (PCL) on their biomechanical properties. After testing the effect of molecular weight (MW), temperature, and humidity on the tensile properties in dry, wet at 25 degrees C and wet at 37 degrees C conditions, chitosan with a MW>310 kD was selected for use in the blend. Homogeneous blends of 25%, 50% and 75% PCL compositions were formed by dissolving chitosan and 80 kD PCL in a common solvent of approximately 77% aqueous acetic acid. Taking advantage of the low melting point of PCL, blend membranes were processed at 25, 37, 55 degrees C water bath or 55 degrees C oven into films. Also, membranes were solvent annealed using chloroform vapors. Tensile properties were analyzed in wet conditions at 25 degrees C. Support for cell viability and distribution of cytoskeletal actin were analyzed by in vitro cell culture of mouse embryonic fibroblasts (MEFs). Differential scanning calorimetry studies indicated the miscibility of the two components when approximated using Nishi-Wang equation. Drying the films at 55 degrees C in an oven formed membranes without separation of two phases. However, the analyzed tensile properties showed no significant alterations relative to chitosan. On the contrary, significant improvements were observed after solvent annealing. Interestingly, increased viability and redistribution of actin fibers was observed on blends formed with 50% PCL and 75% PCL relative to individual polymers. In summary, 50:50 blends when processed at 55 degrees C in an oven showed significant improvement in mechanical properties as well as support for cellular activity relative to chitosan.

Animals↗

PubMed2005

In vitro characterization of chitosan-gelatin scaffolds for tissue engineering.

Recently, chitosan-gelatin scaffolds have gained much attention in various tissue engineering applications. However, the underlying cell-matrix interactions remain unclear in addition to the scaffold degradation and mechanical characteristics. In this study, we evaluated (i) the degradation kinetics of chitosan and chitosan-gelatin scaffolds in the presence of 10mg/L of lysozyme for dimensional stability, weight loss, and pH changes for a period of 2 months, (ii) tensile and compressive properties of films and scaffolds in wet state at 37 degrees C, (iii) viability of fibroblasts and human umbilical vein endothelial cells (HUVECs) on scaffolds, and (iv) the alteration in cell spreading characteristics, cytoskeletal actin distribution, focal adhesion kinase (FAK) distribution and PECAM-1 expression of HUVECs under static and 4.5, 8.5, 13 and 18 dyn/cm2 shear stress conditions. Degradation results showed that gelatin-containing chitosan scaffolds had faster degradation rate and significant loss of material than chitosan. Mechanical properties of chitosan are affected by the addition of gelatin although there was no clear trend. Three-dimensional chitosan and chitosan-gelatin scaffolds supported fibroblast viability equally. However, chitosan membranes decreased cell-spreading area, disrupted F-actin and localized FAK in the nucleus of HUVECs. Importantly, the lowest shear stress tested (4.5 dyn/cm2) for 3 h washed away cells on chitosan suggesting weak cell adhesion. In the blends, effect of gelatin was dominant; actin and FAK distribution were comparable to gelatin in static culture. However, at higher shear stresses, presence of chitosan inhibited shear-induced increase in cell spreading and weakened cell adhesive strength. No significant differences were observed in PECAM-1 expression. In summary, these results showed significant influence of blending gelatin with chitosan on scaffold properties and cellular behavior.

Actins↗

PubMed2002

Interferon gamma modulates trauma-induced muscle wasting and immune dysfunction.

OBJECTIVE: To test the effect of burn injury in mice congenitally deficient in interferon gamma (IFN-gamma) and as well as in wild-type animals treated with IFN-gamma neutralizing antibody. SUMMARY BACKGROUND DATA: The mechanisms underlying muscle wasting following burn trauma are incompletely characterized, although the hypercatabolic state is a consequence of increased proteasomal degradation. Concurrently, burn injury results in an immunocompromised state, and subsequent infections are the leading cause of morbidity and mortality in these patients. IFN-gamma, best conceptualized as a macrophage activating protein, modulates a variety of biologic pathways potentially relevant to muscle wasting and immune dysfunction. METHODS: Mice received either a 20% total body surface area burn or a control sham treatment. At days 1, 2, and 7 following treatment, skeletal muscle, peripheral blood, and spleen were harvested from both groups. Protein synthesis and degradation rates were measured. Lymphocyte subpopulation expression of major histocompatibility complex I (MHC I) molecules was assessed by flow cytometry, and proliferation capacity was measured using mixed lymphocyte reaction. RESULTS: IFN-gamma is critically involved in burn-induced weight loss; moreover, absence of IFN-gamma virtually abolished skeletal muscle hypercatabolism following burn injury. Lymphocyte proliferation and MHC I expression in the setting of burn trauma are also normalized in the absence of IFN-gamma. Both antigen presentation and proliferation functions are independently affected. CONCLUSIONS: IFN-gamma plays a fundamental role in mediating the hypercatabolic state of multiple cell types following burn trauma.

Animals↗

PubMedDate not supplied

Modeling the inhibitor activity and relative binding affinities in enzyme-inhibitor-protein systems: application to developmental regulation in a PG-PGIP system.

Within a number of classes of hydrolytic enzymes are certain enzymes whose activity is modulated by a specific inhibitor-protein that binds to the enzyme and forms an inactive complex. One unit of a specific inhibitor-protein activity is often defined as the amount necessary to inhibit one unit of its target enzyme by 50 %. No objective quantitative means is available to determine this point of 50 % inhibition in crude systems such as those encountered during purification. Two models were derived: the first model is based on an irreversible binding approximation, and the second, or equilibrium, model is based on reversible binding. The two models were validated using the inhibition data for the polygalacturonase-polygalacturonase-inhibiting protein (PG-PGIP) system. Theory and experimental results indicate that the first model can be used for inhibitor protein activity determination and the second model can be used for inhibitor protein activity determination as well as for comparison of association constants among enzymes and their inhibitor-proteins from multiple sources. The models were used to identify and further clarify the nature of a differential regulation of expression of polygalacturonase-inhibiting protein in developing cantaloupe fruit. These are the first relations that provide for an objective and quantitative determination of inhibitor-protein activity in both pure and crude systems. Application of these models should prove valuable in gaining insights into regulatory mechanisms and enzyme-inhibitor-protein interactions.

Computer Simulation↗