Vignesh Muthuvijayan

Analysis of functionalized polyethylene terephthalate with immobilized NTPDase and cysteine.

Polyethylene terephthalate (PET) was functionalized to introduce carboxyl groups onto its surface by a carboxylation technique. Surface and bulk properties, such as possible surface deterioration, surface roughness and the mechanical strength of the carboxylated polymers, were studied and compared with those of aminolyzed and hydrolyzed PET. Atomic force microscopy studies showed that unlike aminolysis and hydrolysis, which increased the surface roughness significantly due to cracking and pitting, the surface roughness of unmodified and carboxylated PET were comparable. While hydrolysis and aminolysis of PET resulted in significant loss of strength, tensile testing revealed that unmodified and carboxylated polymers had similar strength. The development of mechanically stable, functionalized PET would vastly improve the biomedical applications of this polymer. To understand the potential for improving biomedical applications, biologically active molecules, namely nucleoside triphosphate diphosphohydrolase (NTPDase) and cysteine, were immobilized on the carboxylated PET using amide bonds. NTPDase was also immobilized to aminolyzed PET using imine bonds, while cysteine was immobilized on aminolyzed PET using both imine and amide bonds. Attachment of NTPDase and cysteine was verified by analyzing the NTPDase activity and the cysteine surface concentration. The stability of these immobilizations was also studied.

Fabrication of chitosan/gallic acid 3D microporous scaffold for tissue engineering applications

This study explores the potential of gallic acid incorporated chitosan (CS/GA) 3D scaffolds for tissue engineering applications. Scaffolds were prepared by freezing and lyophilization technique and characterized. FTIR spectra confirmed the presence of GA in chitosan (CS) gel. DSC and TGA analysis revealed that the structure of chitosan was not altered due to the incorporation of GA, but thermal stability was significantly increased compared to the CS scaffold. SEM micrographs showed smooth, homogeneous, and microporous architecture of the scaffolds with good interconnectivity. CS/GA scaffolds exhibited approximately 90% porosity on average, increased swelling (600-900%) and controlled biodegradation (15-40%) in PBS (pH 7.4 at 37°C) with 1 mg/mL of lysozyme. CS/GA scaffolds showed 2-4 fold decrease in CFUs (p < 0.05) for both gram positive and gram negative bacteria compared to the CS scaffold. Cytotoxicity of these scaffolds was evaluated using NIH 3T3 L1 fibroblast cells. CS/GA 0.25% scaffold showed similar viability with CS scaffold at 24 and 48 h. CS/GA scaffolds (0.5-1.0%) showed 60-75% viability at 24 h and 90% at 48 h. SEM images showed that an increased cell attachment was observed for CS/GA scaffolds compared to CS scaffolds. These findings authenticate that CS/GA scaffolds were cytocompatible and would be useful for tissue engineering applications.

Differential Adhesive and Bioactive Properties of the Polymeric Surface Coated with Graphene Oxide Thin Film

Surface engineering of implantable devices involving polymeric biomaterials has become an essential aspect for medical implants. A surface enhancement technique can provide an array of unique surface properties that improve its biocompatibility and functionality as an implant. Polyurethane-based implants that have found extensively acclaimed usage as an implant in biomedical applications, especially in the area of cardiovascular devices, still lack any mechanism to ward off bacterial or platelet adhesion. To bring out such a defense mechanism we are proposing a surface modification technique. Graphene oxide (GO) in very thin film form was wrapped onto the electrospun fibroporous polycarbonate urethane (PCU) membrane (GOPCU) by a simple method of electrospraying. In the present study, we have developed a simple single-step method for coating a polymeric substrate with a thin GO film and evaluated the novel antiadhesive activity of these films. SEM micrographs after coating showed the presence of very thin GO films over the PCU membrane. On the GOPCU surface, the contact angle was shifted by ∼30°, making the hydrophobic PCU surface slightly hydrophilic, while Raman spectral characterization and mapping showed the presence and distribution of GO over 75% of the membrane. A reduced platelet adhesion on the GOPCU surface was observed; meanwhile, bacterial adhesion also got reduced by 85% for Staphylococcus aureus (Gram positive, cocci) and 64% for Pseudomonas aeruginosa (Gram negative, bacilli). A cell adhesion study conducted using mammalian fibroblast cells projected its proliferation percentage in a MTT assay, with 82% cell survival on PCU and 86% on GOPCU after 24 h culture, while a study for an extended period of 72 h showed 87% of survival on PCU and 88% on GOPCU. This plethora of functionalities by a simple modification technique makes thin GO films a self-sufficient surface engineering material for future biomedical applications.

Biomimetic hydrogel loaded with silk and l‐proline for tissue engineering and wound healing applications

The aim of this article was to develop silk protein (SF) and l-proline (LP) loaded chitosan-(CS) based hydrogels via physical cross linking for tissue engineering and wound healing applications. Silk fibroin, a biodegradable and biocompatible protein, and l-proline, an important imino acid that is required for collagen synthesis, were added to chitosan to improve the wound healing properties of the hydrogel. Characterization of these hydrogels revealed that CS/SF/LP hydrogels were blended properly and LP incorporated hydrogels showed excellent thermal stability and good surface morphology. Swelling study showed the water holding efficiency of the hydrogels to provide enough moisture at the wound surface. In vitro biodegradation results demonstrated that the hydrogels had good degradation rate in PBS with lysozyme. LP loaded hydrogels showed approximately a twofold increase in antioxidant activity. In vitro cytocompatibility studies using NIH 3T3 L1 cells showed increased cell viability (p < 0.01), migration, proliferation and wound healing activity (p < 0.001) in LP loaded hydrogels compared to CS and CS/SF hydrogels. Cell adhesion on SF and LP hydrogels were observed using SEM and compared to CS hydrogel. LP incorporation showed 74-78% of wound closure compared to 35% for CS/SF and 3% for CS hydrogels at 48 h. These results suggest that incorporation of LP can significantly accelerate wound healing process compared to pure CS and SF-loaded CS hydrogels. Hence, CS/LP hydrogels could be a potential wound dressing material for the enhanced wound tissue regeneration and repair.

Accelerated Healing of Diabetic Wounds Treated with L-Glutamic acid Loaded Hydrogels Through Enhanced Collagen Deposition and Angiogenesis: An In Vivo Study

We have developed L-glutamic acid (LG) loaded chitosan (CS) hydrogels to treat diabetic wounds. Although literature reports wound healing effects of poly(glutamic acid)-based materials, there are no studies on the potential of L-glutamic acid in treating diabetic wounds. As LG is a direct precursor for proline synthesis, which is crucial for collagen synthesis, we have prepared CS + LG hydrogels to accelerate diabetic wound healing. Physiochemical properties of the CS + LG hydrogels showed good swelling, thermal stability, smooth surface morphology, and controlled biodegradation. The addition of LG to CS hydrogels did not alter their biocompatibility significantly. CS + LG hydrogel treatment showed rapid wound contraction compared to control and chitosan hydrogel. Period of epithelialization is significantly reduced in CS + LG hydrogel treated wounds (16 days) compared to CS hydrogel (20 days), and control (26 days). Collagen synthesis and crosslinking are also significantly improved in CS + LG hydrogel treated diabetic rats. Histopathology and immunohistochemistry results revealed that the CS + LG hydrogel dressing accelerated vascularization and macrophage recruitment to enhance diabetic wound healing. These results demonstrate that incorporation of LG can improve collagen deposition, and vascularization, and aid in faster tissue regeneration. Therefore, CS + LG hydrogels could be an effective wound dressing used to treat diabetic wounds.

Development of reduced graphene oxide (rGO)-isabgol nanocomposite dressings for enhanced vascularization and accelerated wound healing in normal and diabetic rats

Treatment of chronic non-healing wounds in diabetes is still a major clinical challenge. Here, we have developed reduced graphene oxide (rGO) loaded isabgol nanocomposite scaffolds (Isab + rGO) to treat normal and diabetic wounds. rGO was synthesized by rapid reduction of graphene oxide (GO) under focused solar radiation. Then, rGO was uniformly dispersed into isabgol solution to prepare Isab + rGO nanocomposite scaffolds. These scaffolds were characterized using various physiochemical techniques. Isab + rGO nanocomposite scaffolds showed suitable cell viability, proliferation, and attachment. In vivo experiments were performed using Wistar rats to study the wound healing efficacy of these scaffolds in normal and diabetic rats. Results revealed that rGO stimulated collagen synthesis, collagen crosslinking, wound contraction, and reduced the wound re-epithelialization time significantly compared to control. Histology and immunohistochemistry analyses showed that Isab + rGO scaffold treatment enhanced angiogenesis, collagen synthesis, and deposition in treated wounds. Isab + rGO scaffold treatment also played a major role in shortening the inflammation phase and recruiting macrophages to enhance the early phase of wound healing. Overall, this investigation showed that Isab + rGO scaffold dressing could significantly accelerate the healing of normal and diabetic wounds.

Isabgol–silk fibroin 3D composite scaffolds as an effective dermal substitute for cutaneous wound healing in rats

Composite 3D scaffolds using a natural, carbohydrate polymer, isabgol (Isab) and silk fibroin (SF) were prepared by a freeze drying method. 2 wt% solutions of both Isab and SF were blended in four different ratios to obtain the 3D scaffolds. ATR-FTIR results showed the presence of amide bonds and β sheet conformation in Isab/SF scaffolds. SEM micrographs showed the fibrous foam like architecture in the Isab/SF blend. Scaffolds showed sufficient porosity and absorbed higher amounts of PBS solution, enhancing the gas and nutrient supply to the cells. Subsequently, scaffolds showed significant in vitro biodegradation. In vitro cytocompatibility test in NIH 3T3 fibroblast cells showed better viability, proliferation and cell attachment for the Isab/SF 75/25 scaffolds compared to the control. Among all the scaffolds, Isab/SF 75/25 showed a faster wound contraction rate (p < 0.001) and reduced period of epithelialization (16 days) compared to the control (23 days). Shrinkage temperature and collagen content was also increased significantly (p < 0.001) in Isab/SF 75/25 scaffold treated rats compared to the control. Histopathology results strongly demonstrate higher cellular infiltration, increased fibroblasts, dense collagen fibers, and neovascularization in Isab/SF 75/25 treated rats, compared to other treatments. Overall, these results strongly authenticate that the Isab/SF 75/25 3D composite scaffolds enhanced the fluid uptake ability with enough fibroblast attachment and good viability. Also, it accelerated tissue regeneration during wound healing in rats without addition of any bioactive molecules.

Morin incorporated polysaccharide–protein (psyllium–keratin) hydrogel scaffolds accelerate diabetic wound healing in Wistar rats

Chronic wounds cost several billion dollars of public healthcare spending annually and continue to be a persistent threat globally. Several treatment methods have been explored, and all of them involve covering up the wound with therapeutic dressings that reduce inflammation and accelerate the healing process. In this present study, morin (MOR) was loaded onto hydrogel scaffolds prepared from psyllium seed husk polysaccharide (PSH), and human hair keratins (KER) crosslinked with sodium trimetaphosphate. ATR-FTIR confirmed the presence of the constituent chemical ingredients. SEM images of the scaffold surface reveal a highly porous architecture, with about 80% porosity measured by liquid displacement measurement, irrespective of the morin concentration. Swelling assays carried out on the scaffolds portray an ability to absorb up to seven times their dry weight of fluids. This makes them attractive for guiding moist wound healing on medium exuding wounds. An Alamar blue assay of NIH/3T3 fibroblast cells shows that cell viability decreases in the first 24 h but recovers to 85% in comparison to a control after 48 h. SEM images of fibroblast cells grown on the scaffolds confirm cellular attachment. An in vivo diabetic wound healing study showed that PSH + KER + MOR scaffold treatment significantly reduced the re-epithelialization time (p < 0.01) and enhanced the rate of wound contraction (p < 0.001), by accelerating collagen synthesis in diabetic rats compared to controls.

An investigation of konjac glucomannan-keratin hydrogel scaffold loaded with Avena sativa extracts for diabetic wound healing

We have developed a novel hydrogel composed of konjac glucomannan (KGM), human hair proteins (KER), and an ethanolic extract of Avena sativa (OAT) and evaluated its potential as a dressing material for diabetic wounds. KGM is an excellent biocompatible gelling agent that stimulates fibroblast proliferation and immunomodulation. Human hair proteins (KER) are biocompatible, biodegradable, and possess abundant cell adhesion sites. KER also promotes fibroblast attachment and proliferation, keratinocyte migration, and collagen expression, which can accelerate wound healing. OAT consists of oat β-glucans and several anti-inflammatory and antioxidant moieties that can reduce prolonged inflammation in chronic wounds. SEM images confirm the highly porous architecture of the scaffolds. When immersed in PBS, KGM+KER+OAT hydrogels absorb 7.5 times their dry weight. These hydrogels display a measured rate of degradation in lysozyme. KGM+KER+OAT hydrogels showed no significant cytotoxicity against NIH/3T3 fibroblasts. DAPI and SEM images obtained after 48h of cell culture illustrate the attachment and infiltration of fibroblasts. In vivo studies performed using a diabetic rat excision wound model showed that KGM+KER+OAT hydrogels significantly accelerated wound healing compared to the control and the KGM+KER hydrogels.

Reduced graphene oxide-loaded nanocomposite scaffolds for enhancing angiogenesis in tissue engineering applications

Tissue engineering combines cells, scaffolds and signalling molecules to synthesize tissues in vitro. However, the lack of a functioning vascular network severely limits the effective size of a tissue-engineered construct. In this work, we have assessed the potential of reduced graphene oxide (rGO), a non-protein pro-angiogenic moiety, for enhancing angiogenesis in tissue engineering applications. Polyvinyl alcohol/carboxymethyl cellulose (PVA/CMC) scaffolds loaded with different concentrations of rGO nanoparticles were synthesized via lyophilization. Characterization of these scaffolds showed that the rGO-loaded scaffolds retained the thermal and physical properties (swelling, porosity and in vitro biodegradation) of pure PVA/CMC scaffolds. In vitro cytotoxicity studies, using three different cell lines, confirmed that the scaffolds are biocompatible. The scaffolds containing 0.005 and 0.0075% rGO enhanced the proliferation of endothelial cells (EA.hy926) in vitro. In vivo studies using the chick chorioallantoic membrane model showed that the presence of rGO in the PVA/CMC scaffolds significantly enhanced angiogenesis and arteriogenesis.