Name: doxycycline loaded collagen-chitosan composite scaffold for the accelerated healing of diabetic wounds
Uploaded: 2021-08-21T14:00:00
Description: Watch this Scientific Journal Video about Doxycycline Loaded Collagen-Chitosan Composite Scaffold for the Accelerated Healing of Diabetic Wounds at JoVE.com

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&#160;M. Pharm students for their support in the video shoot.
This research was supported by the JSS Academy of Higher Education &#38; Research (JSSAHER).

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

<ol>
	<li>. IDF Diabetes Atlas, 9th edn Available from: <a target="_blank" href="https://www.diabetesatlas.org">https://www.diabetesatlas.org</a> (2019)</li><li>Falanga, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+and+its+impairment+in+the+diabetic+foot.">Wound healing and its impairment in the diabetic foot.</a> The Lancet. 366 (9498), 1736-1743 (2005).</li><li>Frykberg, R. 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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...

Diabetes mellitus (DM) is a condition where the body&#39;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.
Plenty of treatments, such as instructing the patients (inspect wound daily, cleanse the wound, avoid activities that creates pressure on the wound, periodic glucose monitoring, etc.), controlling their blood glucose, wound debridement, pressure offloading, medical procedure, hyperbaric oxygen therapy, and advanced therapies are in practice&#160;3,4. The majority of these medications fail to address all prerequisites vital for DW care in light of the multifactorial pathophysiological conditions and unexpected expenses related to these medicines&#160;5. Even though the DW pathogenesis is multifactorial, the persistent inflammation with inappropriate tissue management is stated to be the actual reason for delayed healing in DWs 5,6.
Augmented levels of inflammatory and pro-inflammatory mediators in DW result in diminished growth factors responsible for delayed wound healing 2,6. Improper extracellular matrix (ECM) formation in DWs is accredited to increased levels of matrix metalloproteinases (MMPs) accountable for the rapid degradation of formed ECM. In MMPs, MMP-9 is reported as a major intermediary responsible for prolonged inflammation and rapid ECM degradation 7. It is stated that local treatment with an anti-inflammatory drug that decreases the elevated levels of MMP-9 re-establishes cutaneous homeostasis, framework arrangement and prompts better healing of DWs 8,9.
Doxycycline (DOX), an MMP-9 inhibitor, was chosen to suppress the elevated levels of MMP-9, a major inflammatory mediator responsible for persistent inflammation in DWs 10,11,12. In addition, DOX possess antioxidant (produce free hydroxy and phenoxy radicals capable of binding with reactive oxygen species) 13 and anti-apoptotic (inhibit caspase expression and mitochondrial stabilization) 14 activities that are essential for the treatment of DW. The arrangement of frameworks containing DOX, collagen (COL), and chitosan (CS) was chosen. The choice of COL depends on the way that it helps in providing the necessary framework responsible for mechanical strength and tissue regeneration 15. On the other hand, CS is structurally homologous to glycosaminoglycan, associated with several wound healing phases. It is also reported that CS holds significant anti-bacterial property 15. Hence, the COL/CS scaffold of DOX is formulated to suppress the prolonged inflammation, followed by supporting the matrix formation for successful wound healing in DM conditions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-ethyl-(3-3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC)</td>
			<td>Merck Millipore, Mumbai, India</td>
			<td>E7750</td>
			<td></td>
		</tr>
		<tr>
			<td>2-(N-morpholino) ethane sulfonic acid (MES)</td>
			<td>Merck Millipore, Mumbai, India</td>
			<td>137074</td>
			<td></td>
		</tr>
		<tr>
			<td>3-(4, 5 dimethyl thiazole-2 yl) -2, 5-diphenyl tetrazolium bromide (MTT)</td>
			<td>Thermo Fisher, Mumbai, India</td>
			<td>M6494</td>
			<td></td>
		</tr>
		<tr>
			<td>Deep freezer verticle</td>
			<td>Labline Instruments, Kochi, India</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis sack</td>
			<td>Merck Millipore, Mumbai, India</td>
			<td>D6191-Avg. flat width 25 mm (1.0 in.), MWCO 12,000 Da</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Sigma chemicals Co. Ltd, Mumbai, India</td>
			<td>D9891</td>
			<td></td>
		</tr>
		<tr>
			<td>Elisa kit</td>
			<td>R&#38;D Systems</td>
			<td>RMP900</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli (E. coli)</td>
			<td>National Collection of Industrial Microorganisms, Pune, India</td>
			<td>NCIM 2567</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck Millipore, Mumbai, India</td>
			<td>100983</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyophilizer-SZ042</td>
			<td>Sub-Zero lab instruments, Chennai, India</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mechanical Stirrer-RQ-122/D</td>
			<td>Remi laboratory instruments, Mumbai, India</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medium molecular weight Chitosan</td>
			<td>Sisco Research Laboratories Pvt. Ltd., Mumbai, India</td>
			<td>18824</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome-RM2135</td>
			<td>Leica, U.K</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse embryonic fibroblast cells (3T3-L1)</td>
			<td>National Centre for Cell Sciences, Pune, India</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multiple plate reader -Inifinte M200 Pro</td>
			<td>Tecan Instruments, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N-hydroxy succinimide (NHS)</td>
			<td>Sigma chemicals Co. Ltd, Mumbai, India</td>
			<td>130672</td>
			<td></td>
		</tr>
		<tr>
			<td>Pseudomonas aeruginosa (P. aeruginosa)</td>
			<td>National Collection of Industrial Microorganisms, Pune, India</td>
			<td>NCIM 2036</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscopy (SEM)-S-4800</td>
			<td>Hitachi, India</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH) pellets</td>
			<td>Qualigen fine chemicals, Mumbai, India</td>
			<td>Q27815</td>
			<td></td>
		</tr>
		<tr>
			<td>Staphylococcus aureus (S. aureus)</td>
			<td>National Collection of Industrial Microorganisms, Pune, India</td>
			<td>NCIM 5022</td>
			<td></td>
		</tr>
		<tr>
			<td>Staphylococcus epidermis (S. epidermis)</td>
			<td>National Collection of Industrial Microorganisms, Pune, India</td>
			<td>NCIM 5270</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptozotocin (STZ)</td>
			<td>Sisco Research Laboratories Pvt. Ltd., Mumbai, India</td>
			<td>14653</td>
			<td></td>
		</tr>
		<tr>
			<td>Type-1 rat Collagen</td>
			<td>Sigma chemicals Co. Ltd, Mumbai, India</td>
			<td>C7661</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraviolet&#8211;visible spectroscopy-1700</td>
			<td>Shimadzu</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

doxycycline loaded collagen-chitosan composite scaffold for the accelerated healing of diabetic wounds

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) &amp; 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 &amp; 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&#8804;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.

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 ...

Watch this Scientific Journal Video about Doxycycline Loaded Collagen-Chitosan Composite Scaffold for the Accelerated Healing of Diabetic Wounds at JoVE.com

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

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 ...

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