N. Shanmugasundaram

In vivo and in vitro evaluation of hair growth potential of Hibiscus rosa-sinensis Linn.

Petroleum ether extract of leaves and flowers of Hibiscus rosa-sinensis was evaluated for its potential on hair growth by in vivo and in vitro methods. In vivo, 1% extract of leaves and flowers in liquid paraffin was applied topically over the shaved skin of albino rats and monitored and assessed for 30 days. The length of hair and the different cyclic phases of hair follicles, like anagen and telogen phases, were determined at different time periods. In vitro, the hair follicles from albino rat neonates were isolated and cultured in DMEM supplemented with 0.01 mg/ml petroleum ether extract of leaves and flowers. From the study it is concluded that the leaf extract, when compared to flower extract, exhibits more potency on hair growth.

Gelatin microspheres cross-linked with EDC as a drug delivery system for doxycyline : Development and characterization

Chronic wounds express elevated levels of proteases, in particular matrix metalloproteinases (MMPs), which degrades de novo granulation tissue and endogenous biologically active proteins. An effective therapeutic approach for chronic wounds would be to modify this hostile environment and reduce the proteolytic imbalance. Doxycycline has been proved recently to inhibit MMPs and used topically for chronic wound ulcers, beyond their antimicrobial profile. To this end, a carrier system for controlled release of doxycycline, suitable for incorporation into various wound dressings like membranes and sponges was developed. In the present study gelatin microspheres, cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was proposed. The cross-linking was carried out with different concentrations of EDC (10 mM, 50 mM and 100 mM) and for different time periods (3–24 h). The cross-linked microspheres were characterized by evaluating the extent of cross-linking, the morphology, swelling behaviour and drug loading and in vitro studies of drug release, enzymatic degradation and biocompatibility. The extent of cross-linking increased as a function of both EDC concentration and the cross-linking time periods. It is found that the extent of cross-linking greatly influences the swelling and drug release behaviour of the microspheres. The cross-linked microspheres were found to be biocompatible to NIH 3T3 mouse embryonic fibroblast. The overall study indicates that the zero length cross-linker EDC can be considered as a potential alternative for cross-linking the gelatin microspheres.

Functionally modified gelatin microspheres impregnated collagen scaffold as novel wound dressing to attenuate the proteases and bacterial growth.

An attempt was made to develop a new therapeutic delivery system which would play a dual role of attenuating MMP activity in the wounds and also prevent infection by controlled delivery of antimicrobials. A catechol type MMP inhibitor 2,3-dihydroxybenzoic acid (DHBA) was conjugated to gelatin microspheres using EDC/NHS as coupling agents. The potential of the modified gelatin microspheres (DHB-MS) to attenuate the proteases such as MMP 2 and MMP 9 in the diabetic wound tissues was investigated by gelatin zymography. Further the modified microspheres were loaded with doxycycline and impregnated in a reconstituted collagen scaffold as novel wound dressing. The in vitro release behavior of doxycycline from both DHB-MS and DHB-MS impregnated collagen scaffold was investigated. DHB-MS when incubated with the tissue lysate for 6h displayed the complete inhibition of the MMPs in the tissue lysate. The in vitro drug release studies from the collagen scaffold exhibited the burst release of 44%, exerted immediate chemo prophylaxis and sustained delivery for 72 h. The MTT assay and fluorescent labeling with calcein AM indicated that the DHB-MS is biocompatible to human foreskin fibroblasts. Thus the system developed provides wider scope to control the pathogens involved in infection and also the excess matrix degradation.

Efficiency of controlled topical delivery of silver sulfadiazine in infected burn wounds

The present study is designed to assess the potential benefits of controlled delivery of silver sulfadiazine from collagen scaffold (SSDM-CS) in infected deep partial thickness burn wounds in which epidermis is lost completely and the entire papillary dermis and most of the recticular layer of the dermis is lost. Infection induced by inoculating 10(7) colony forming units (cfu) of Pseudomonas aeruginosa caused significant increase in wound size (20%) till day 15, which decreased significantly from day 9 by SSDM-CS treatment, showing complete healing by day 27 (control > or = 37 days). Early subsidence of infection (<10(2) cfu, day 9) by SSDM-CS resulted in faster epidermal resurfacing and fibroplasia, whereas heavy microbial load (>10(7) cfu, day 9) in controls caused severe inflammatory cellular infiltration. Persistent infection triggered early expression of proinflammatory cytokines intereukin-6, intereukin 1-beta, and tumor necrosis factor-alpha, lasting until day 9, whereas cytokine level decreased in SSDM-CS-treated group by day 6. Infection exacerbated expression of active matrix metalloproteinases (MMPs)-2 and -9 in controls (day 15), while SSDM-CS positively modulated MMP-2 and -9 with faster decline in their levels (day 12). Inherent nature of the dressing to maintain drug level at equilibrium therapeutic concentration (51.2 microg/mL) for prolonged time (72 h), below systemic toxic limits (20 microg/dL, serum level), accelerated the magnitude and sequence of reparative events.

Collagen-based wound dressing for doxycycline delivery: in-vivo evaluation in an infected excisional wound model in rats.

Objectives A novel collagen‐based dressing consisting of 2,3‐dihydroxybenzoic‐acid‐modified gelatin microspheres loaded with doxycycline has previously been reported to address both infection and matrix degradation. In the present study the potential benefits of the dressing were investigated in an excisional wound model in rats challenged with Pseudomonas aeruginosa.

Comparative Physico-chemical and in Vitro Properties of Fibrillated Collagen Scaffolds from Different Sources

Collagen from different sources was isolated and designed as scaffolds to act as a three-dimensional substrate for culturing human skin fibroblasts, which can be used as dermal substitutes. The thermodynamic behavior of the scaffolds developed was analyzed through Differential Scanning Calorimetric (DSC) and Thermogravimetric analysis (TGA). Analysis by Fourier Transform Infrared Spectroscopy (FTIR) revealed the functional groups in the scaffolds and the mechanical stability of various scaffolds was assessed through tensile strength analysis. Human skin fibroblasts were cultured on the developed scaffolds to assess their cellular interaction and behavior, and the morphological characteristics of the cultured fibroblasts were evaluated using Scanning Electron Microscopy (SEM). The collagen scaffold exhibited unique features when developed from various sources and it was observed that cells could grow and proliferate well and spread as a monolayer in the reconstituted collagen scaffold.

Controlled release of 2, 3 desulfated heparin exerts its anti-inflammatory activity by effectively inhibiting E-selectin

Control of inflammation using appropriate anti-inflammatory agent prevents wound from becoming chronic. Heparin is a heterogeneous mixture of polysaccharide molecules with a mean molecular weight between 12-30 kDa containing 200-300 disaccharide units of glycosaminoglycan chains. Chemical modifications leading to generation of a unique pentasaccharide sequence effectively reduces its anticoagualant activity, while retaining its anti-inflammmatory property. In this study, Standard heparin was partially desulfated to 2, 3 desulfated heparin (2, 3 DSH) and its anti-inflammatory property was determined by assessing its ability to prevent static adhesion of leukocytes to endothelium and clotting assay. The effectiveness of 2, 3 DSH to down regulate E-selectin and key proinflammatory cytokines (IL-1beta and IL-6) was assessed by western blot and immunocytochemistry. These results were compared with commercially available 2-Desulfated Heparin (2DSH) and standard heparin (SH). Finally, a controlled delivery system of 2, 3 DSH was designed using chitosan microspheres and collagen as scaffold. Optimal loading of 2, 3 DSH was achieved and the release kinetics were tuned to follow a controlled release pattern. The steady state concentration of 2, 3 DSH was found to be optimal to elicit anti-inflammatory property and could achieve inhibition of E-selectin expression while unaffecting the normal clotting cascade.

Efficacy of desulfated heparin mitigating inflammation in rat burn wound model

During skin repair, leukocyte infiltration is the principal inflammatory response which is instrumental in triggering growth factor and cytokine signals that orchestrate together to recruit cells necessary for healing. In severe wounds like burn, when acute inflammation becomes chronic, intervention is required to control inflammation so as to hasten the process of healing. Heparin, a known anticoagulant also possesses anti-inflammatory activity by its ability to interfere with the adhesion of leukocytes to the endothelium. Desulfated heparins (DSH) have subdued anticoagulant activity while possessing increased anti-inflammatory activity. Among which 2,3 DSH is found to have marked potency as an anti-inflammatory agent and has been utilized for this study. In this investigation, a controlled delivery system was designed by incorporating 2,3 DSH in microspheres and embedding in collagen matrix which could serve as a wound dressing in burns. In vivo evaluation of healing process was ascertained in rat burn wound model by qualitative and quantitative estimation of proinflammatory cytokines in serum and granulation tissue and collagen turnover was also assessed as healing progressed. The results of this study suggests that 2,3 DSH could be delivered in a controlled manner to regulate inflammatory events to hasten healing of burn wounds.

Inhibition of E-Selectin by Selectively deSulfated Heparin through Chitosan Microspheres Impregnated Collagen Scaffold

Wound healing is a collaborative process involving a variety of cellular and matrix components which need to interact continually towards a common goal. Growth factors and cytokines are two distinct categories of signaling proteins that modulate wound healing at molecular and cellular levels. Cells synthesize different proteins for their proliferation and migration which are controlled in a phased manner. Chronic wounds lead to changes in local regulation of inflammation and trigger the adverse effects. It is necessary to control the inflammation through proper agents. Heparin has an anti-inflammatory property when it is selectively desulfated, in dose dependent manner by blocking the extravasation of leukocytes to the endothelium of an injured site. This partially desulfated heparin (DSH) was entrapped in chitosan microspheres developed by novel water in oil emulsion technique and impregnated in collagen scaffold for its controlled delivery. Hence, structurally modified heparins which possess less anti coagulant activity and more anti inflammatory activity were investigated. The concentration of 2, 3 DSH to be used to serve as an anti inflammatory agent was determined using clotting assay and static adhesion of leukocytes to endothelium. The down regulation of E-selectin was assessed by western blot and immunocytochemistry using monoclonal antibodies. The delivery system for controlled release of 2, 3 DSH was designed using chitosan microspheres and collagen as scaffold. The scaffold was characterized for its physicochemical and biological properties. Wound healing studies in rat burn wound model exhibited a faster healing rate in treated group when compared to that of controls. In vitro and in vivo studies support the efficacy of the modified heparin namely 2, 3 DSH as a therapeutic agent to regulate inflammation.

PROGRAMMED CELL DEATH OF HUMAN SKIN FIBROBLAST INDUCED BY BENZIMIDAZOLE

INTRODUCTION. During the course of apoptosis, several changes occur at cellular and molecular level, among which the superstructural modifications of DNA cleavage and chromatin were studied by DNA laddering assays, transmission electron microscope (TEM) and flow cytometric methods. The present study is focused on the programmed cell death of human skin fibroblast induced by benzimidazole, a widely used fungicide in the tanning industry. MATERIALS AND METHODS. Human skin fibroblasts were maintained at 37 0 C in a humidified incubator in DMEM supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum. Cells were passaged at confluence by trypsinization, with all experiments performed on cells at low passage (≤4). Programmed cell death induced by different concentrations of benzimidazole on cultured skin fibroblast were analyzed by DNA laddering techniques (1), flow cytometry, and TEM analysis was carried out by standard procedures given by Goping et al. (2) and Willingham (3,4). Viability of cells cultured in the 96-well plates was assessed by mitochondrial dehydrogenase activity using the calorimetric MTT assay (4). RESULTS. The ultrastructural studies reveal that benzimidazole causes pyknosis and kryorrhexis (fragmentation) in the nucleus of skin fibroblast. Application of benzimidazole at 10 µg/ml DMEM medium induced shrinkage in fibroblasts and after 3 hours apoptotic body formation was identified. Microscopic analysis showed nuclear disintegration and chromatin granulation. More evidently the DNA laddering and flow cytometry confirm that higher concentration of benzimidazole induced quantitative variations and breaks in DNA and induce cytotoxicity in fibroblast population. At lower concentration of benzimidazole, vesiculation of endoplasmic reticulum is induced and these vesicles after fusion with plasma membrane gave a characteristic blebbed appearance as revealed by light and electron microscopic studies. DISCUSSION. A common hallmark of apoptosis is DNA fragmentation, which was analyzed by DNA laddering techniques. Morphological characteristics and chromatin cleavage in skin fibroblasts was established by cytochemical and electrophoretic methods and the results showed that benzimidazole has the potential to cleave the DNA and act as a potent toxicant. Thus, fibroblast is not killed by benzimidazole, but is induced to commit suicide or apoptosis at lower concentration. At higher concentration of benzimidazole, the fibroblasts lose the spindle shape and take up a round shape and get detached from the substratum. During this process nuclear and cellular fragmentation were observed. The ultrastructural alteration was