Preparation and characterization of a jelly fig (Ficus awkeotsang Makino) polysaccharide-based bioactive 3D scaffold for improved vascularization and skin tissue engineering applications.

Jelly fig polysaccharides (JFP) were extracted from Ficus awkeotsang Makino achenes. The yield of JFP was approximately 10-15 %. FT-IR spectrum of the extracted JFP confirmed that it was made of low methoxyl pectin (LMP). 3D scaffolds of JFP (JFP scaffold) were fabricated using ionic crosslinking of 2 % (w/v) JFP solution with Ca2+ ions and freeze-drying. The JFP scaffold showed 73.46 ± 1.97 % porosity and a 12-fold swelling capacity. The porous morphology was also observed in SEM micrographs. JFP scaffolds were completely degraded in 14 days when incubated in 1 mg/mL lysozyme solution, compared to the 50 % degradation observed in PBS alone. The antioxidant activity of the JFP and JFP scaffold was approximately 40 %. The hemolytic assay of the JFP scaffold showed <5 % (3.0 ± 0.4) RBC lysis. The cytocompatibility of the JFP scaffold was evaluated using L929 mouse fibroblasts and human dermal fibroblasts (HDF). The in vitro studies using L929 cells showed that the JFP scaffold is cytocompatible. HDF cells cultured in the presence of JFP scaffolds show a higher fold cell viability, proliferation, and migration. Collagen expression and deposition were also studied, and no significant changes occurred with JFP scaffold treatment. In vivo CAM assay showed an increase in the number and thickness of blood vessels by 1.185-fold and 1.19-fold, respectively. These results confirm the angiogenic property of the JFP scaffold. These biocompatible and bioactive properties of the JFP scaffold could be beneficial for tissue engineering and regenerative medicine applications.

Topical administration of pullulan gel accelerates skin tissue regeneration by enhancing collagen synthesis and wound contraction in rats.

Here, we have studied the efficacy of pullulan gel on wound healing by performing biochemical, biophysical and histological investigations. A 2 cm2 open excision wound was made on the dorsum of the rats and topically treated with 500 µL of pullulan gel. The control group was left untreated. The povidone-iodine (PI) ointment treated animals were considered as positive control. The granulation tissues formed were collected at different time point intervals and used for various biochemical, biophysical and histological analyses. Biochemical analyses revealed that pullulan gel significantly (p < 0.001) improved the collagen, hexosamine, protein and DNA content. Biophysical analyses resulted in an increased rate of wound contraction (p < 0.001). The period of epithelialization was shorter (p < 0.001) in pullulan gel treated group (11 days) than control (22 days) and PI group (17 days). Histological evaluation on days 4, 8 and 11 substantiated that pullulan gel treatment improved the wound re-epithelialization, dermal regeneration, blood vessels formation and collagen synthesis than in control and PI groups. Interrupted SDS-PAGE of collagen showed an increase in Type III collagen band evident for the healing potential of pullulan gel. Thus, our results strongly prove that pullulan gel could be a potential wound healing agent.

Design and evaluation of Konjac glucomannan-based bioactive interpenetrating network (IPN) scaffolds for engineering vascularized bone tissues.

Synthetic bone grafts are being developed to overcome the limitations of conventional treatments for bone defects. In this study, we have fabricated bioactive binary and novel ternary interpenetrating polymer network (IPN) scaffolds using a combination of natural and synthetic polymers. The binary IPN scaffolds were prepared using Konjac glucomannan (KGM) and polyvinyl alcohol (PVA). In the novel ternary IPN scaffolds, polycaprolactone (PCL) was added to PVA and KGM. SEM images showed that these scaffolds were microporous with good interconnectivity. Compression testing confirmed that both the scaffolds are mechanically strong, with the ternary scaffolds having moduli comparable to the natural bone. In vitro cytocompatibility studies performed with NIH/3T3 fibroblasts cells and MG-63 osteosarcoma cells demonstrated the non-toxic and osseointegrating nature of the scaffolds. Confocal images confirmed that the cells migrated into the interconnected pores of the scaffolds. RT-PCR analysis showed that both binary and ternary scaffolds enhanced the expression of the major bone marker genes, viz., ALP, BMP-2, COLLAGEN-1, and OSTEOCALCIN. However, the expression of these osteogenic markers was significantly enhanced in the ternary scaffolds compared to the binary scaffolds. In vivo chick chorioallantoic membrane (CAM) assay shows that these scaffolds possess excellent pro-angiogenic properties. Hence, these desirable biological properties, coupled with the suitable physicochemical properties, make these IPN scaffolds ideal for treating bone defects.

Effect of ethanolic extract of Melia dubia leaves on full-thickness cutaneous wounds in Wistar rats.

In recent years, the therapeutic effects of phyto-principles have been appreciated for their promising effects on wound healing. Melia dubia (Malabar neem) possesses anti-cancer, anti-diabetic, anti-tumor, anti-inflammatory, antioxidant, antibacterial, and fungicidal properties. Here, we studied the wound healing efficacy of ethanolic extract of M. dubia leaves on cutaneous wound healing for the first time. The ethanolic extract of M. dubia was applied topically on the wounds of the experimental rats until the wounds heal completely. Wounds of the control rats were treated with PBS. Granulation tissues formed on wound surfaces of the excision wound were harvested on days 4 and 8 and analyzed to determine the total collagen and hexosamine content. Total collagen and hexosamine were significantly (p < .001) higher in M. dubia treated rats compared to control. The rate of wound closure was significantly higher (p < .001) and period of epithelialization was shorter in M. dubia treated rats. Incision wound tissue was used for the tensile strength measurement. Tensile strength was improved in M. dubia treated wound tissues. Results concluded that the topical application of ethanolic extracts of M. dubia improved the rate of wound contraction and tensile strength by increased synthesis of collagen.

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 ß-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.

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.

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.

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. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1401-1408, 2017.

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.