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Bioengineering

Doxycycline Loaded Collagen-Chitosan Composite Scaffold for the Accelerated Healing of Diabetic Wounds

Published: August 21st, 2021

DOI:

10.3791/62184

1Department of Pharmaceutics, JSS College of Pharmacy, JSS Academy of Higher Education & Research, 2Department of Pharmaceutical Sciences, School of Pharmacy, North Dakota state university, 3Department of Pharmacology, JSS College of Pharmacy, JSS Academy of Higher Education & Research

The prepared DOX-CL scaffold satisfied the prerequisites of an ideal DW dressing in mechanical strength, porosity, water absorption, degradation rate, sustained release, anti-bacterial, biocompatibility, and anti-inflammatory properties, which are considered to be essential for the recovery of damaged tissue in DWs.

One major complication of diabetes mellitus is diabetic wounds (DW). The prolonged phase of inflammation in diabetes obstructs the further stages of an injury leading to delayed wound healing. We selected doxycycline (DOX), as a potential drug of choice, due to its anti-bacterial properties along with its reported anti-inflammatory properties. The current study aims to formulate DOX loaded collagen-chitosan non-crosslinked (NCL) & crosslinked (CL) scaffolds and evaluate their healing ability in diabetic conditions. The characterization result of scaffolds reveals that the DOX-CL scaffold holds ideal porosity, a low swelling & degradation rate, and a sustained release of DOX compared to the DOX-NCL scaffold. The in vitro studies reveal that the DOX-CL scaffold was biocompatible and enhanced cell growth compared with CL scaffold treated and control groups. The anti-bacterial studies have shown that the DOX-CL scaffold was more effective than the CL scaffold against the most common bacteria found in DW. Using the streptozotocin and high-fat diet-induced DW model, a significantly (p≤0.05) faster rate of wound contraction in the DOX-CL scaffold treated group was observed compared to those in CL scaffold treated and control groups. The use of the DOX-CL scaffold can prove to be a promising approach for local treatment for DWs.

Diabetes mellitus (DM) is a condition where the body's failure to deliver insulin or react to its outcomes in abnormal digestion of straightforward sugars brings about an upsurge of blood glucose 1. The most consecutive and crushing entanglement of DM is the diabetic wound (DW). Roughly 25% of patients with DM have the opportunity to build up a DW in their lifetime 1. The hindered healing of DW is accredited to a triopathy of DM: immunopathy, vasculopathy, and neuropathy. Whenever DW is left untreated, it may result in gangrene development, therefore prompting the removal of the concerned organ 2

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All the animal procedures performed were approved by the institutional animal ethical committee of JSS College of Pharmacy, Ooty, India.

1. Preparation of DOX loaded porous scaffolds by freeze-drying method

  1. Add 1.2 g of COL to 100 mL of water (e.g., Millipore) and keep aside for swelling.
  2. Stir the swollen COL dispersion at 2000 rpm overnight to ensure complete dissolution of COL.
  3. Prepare CS solution by dissolving approximately 0.8 g of CS in 100 mL of 1% acetic .......

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Characterization of the DOX loaded NCL and CL scaffold
On visual examination, the NCL and CL scaffold was found to be cream in color. Besides, both the scaffolds appear to be like a sponge, stiff and inelastic when examined physically. SEM images of the NCL and CL scaffolds are shown in Figure 1. From the figure, it was clear that there was a decrease in pore size after crosslinking by forming intermolecular linkages. Also, the NCL and CL scaffolds porosity were found .......

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The main objective of this study was to determine the effect of DOX loaded COL-CS scaffold on DW healing in rats. CL and NCL were prepared and evaluated in terms of morphology, swelling index, in vitro release kinetics, and biocompatibility.

Characterization of the DOX loaded NCL and CL scaffold
The prepared scaffolds were found to be porous with interconnected pores. These interconnected pores assure the porous, spongy nature that helps in the proper diffusion of oxygen.......

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The authors thank Dr. Ashish D Wadhwani. (Assistant Professor and Head, Department of Pharmaceutical Biotechnology, JSS College of Pharmacy, Ooty, India) for assisting in In vitro cell viability studies.

The authors would like to thank the Department of Science and Technology - Fund for Improvement of Science and Technology Infrastructure in Universities and Higher Educational Institutions (DST-FIST), New Delhi, for supporting our department.

The authors also like to thank Mr. Sanju. S and Mr. Sriram. Narukulla M. Pharm students for their support in the video shoot.

This ....

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Name Company Catalog Number Comments
1-ethyl-(3-3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) Merck Millipore, Mumbai, India E7750
2-(N-morpholino) ethane sulfonic acid (MES) Merck Millipore, Mumbai, India 137074
3-(4, 5 dimethyl thiazole-2 yl) -2, 5-diphenyl tetrazolium bromide (MTT) Thermo Fisher, Mumbai, India M6494
Deep freezer verticle Labline Instruments, Kochi, India
Dialysis sack Merck Millipore, Mumbai, India D6191-Avg. flat width 25 mm (1.0 in.), MWCO 12,000 Da
Doxycycline Sigma chemicals Co. Ltd, Mumbai, India D9891
Elisa kit R&D Systems RMP900
Escherichia coli (E. coli) National Collection of Industrial Microorganisms, Pune, India NCIM 2567
Ethanol Merck Millipore, Mumbai, India 100983
Lyophilizer-SZ042 Sub-Zero lab instruments, Chennai, India
Mechanical Stirrer-RQ-122/D Remi laboratory instruments, Mumbai, India
Medium molecular weight Chitosan Sisco Research Laboratories Pvt. Ltd., Mumbai, India 18824
Microtome-RM2135 Leica, U.K
Mouse embryonic fibroblast cells (3T3-L1) National Centre for Cell Sciences, Pune, India
Multiple plate reader -Inifinte M200 Pro Tecan Instruments, Switzerland
N-hydroxy succinimide (NHS) Sigma chemicals Co. Ltd, Mumbai, India 130672
Pseudomonas aeruginosa (P. aeruginosa) National Collection of Industrial Microorganisms, Pune, India NCIM 2036
Scanning Electron Microscopy (SEM)-S-4800 Hitachi, India
Sodium hydroxide (NaOH) pellets Qualigen fine chemicals, Mumbai, India Q27815
Staphylococcus aureus (S. aureus) National Collection of Industrial Microorganisms, Pune, India NCIM 5022
Staphylococcus epidermis (S. epidermis) National Collection of Industrial Microorganisms, Pune, India NCIM 5270
Streptozotocin (STZ) Sisco Research Laboratories Pvt. Ltd., Mumbai, India 14653
Type-1 rat Collagen Sigma chemicals Co. Ltd, Mumbai, India C7661
Ultraviolet–visible spectroscopy-1700 Shimadzu

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