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

Summary

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.

Abstract

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.

Introduction

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.

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 ^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 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.

Protocol

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 acid.
4. Stir the CS solution overnight at 2000 rpm to ensure uniform dispersion.
5. Mix DOX (1% w/v), followed by CS solution, to the COL solution, and stir for 30 min.
6. Filter the obtained physical mixture using a muslin cloth to remove the particulate matter.
7. Deep freeze the obtained filtrate at -85 °C ± 4 °C for about 24 h.
8. Lyophilize the deep freeze mixture at -85 °C ± 4 °C for 72 h.
9. Store the obtained scaffolds in a desiccator for further analysis ^16,17.

2. Crosslinking of scaffold

1. Dissolve MES (0.488 g) in 50 mL of water.
2. Soak 50 mg of the DOX loaded scaffold in 20 mL of the MES buffer for 30 min.
3. Mix 19.5 mL of MES buffer with 0.1264 g of EDC and 0.014 g of NHS in a separate beaker.
4. Immerse the scaffold in the buffer mixture for 4 h to achieve crosslinking ¹⁶.
5. Store the DOX loaded crosslinked (CL) and non-crosslinked scaffolds (NCL) for further evaluation.

3. Characterization of scaffolds

1. Morphological examination using a scanning electron microscopy (SEM)
   1. Characterize the scaffolds for morphological analysis using SEM (1 cm × 1 cm × 0.5 cm).
   2. Stain the cross-section and exterior surface of the scaffold with the delicate layer of gold (~150 Å).
   3. Capture the photographic image at the excitation voltage of 5 kV and 10 kV.
   4. Place the samples in aluminum stubs and enclose them with the gold at approximately 9 V.
   5. Measure the scaffold using SEM with the increased resolution at 10 kV.
2. Porosity determination
   1. Measure the porosity of the scaffolds using the liquid displacement method (ethanol) ¹⁸.
   2. Calculate the porosity of the scaffolds using the below formulae.
      
      W_w = Wet weight of the scaffold
      W_d = Dry weight of the scaffold
      W_v = Volume of the scaffold
3. Determining the water absorption capacity
   1. Measure the dry weight of the scaffold.
   2. Incubate the weighed scaffold at 37 °C for 24 h in phosphate buffer saline (PBS) pH 7.4.
   3. Remove the excess PBS over the scaffold using filter paper.
   4. Measure the water absorption capacity using the below formulae ¹⁷.
      
      W_S = Percentage of water absorption
      W₁=Wet weight of the scaffold
      W₀= Dry weight of the scaffold
4. Scaffold degradation
   1. Incubate the scaffold (1cm x 1cm) at 37 °C for 7 days in a PBS of pH 7.4 containing lysozymes.
   2. Wash the scaffold to remove any adhered ions on the surface.
   3. Freeze dry the washed scaffold ¹⁷.
   4. Calculate the rate of degradation using formulae.
      
      Ww = Initial weight of the scaffold
      Wd = Weight of the scaffold after freeze-drying
5. In vitro release studies
   1. Determine the release behavior of the DOX from the scaffold using the dialysis sack method.
   2. Disperse the scaffold in a few milliliters of simulated wound fluid (pH 7.4) and transfer it into a dialysis bag.
   3. Tightly close the ends of the membrane bag and immerse in the 500 mL of simulated wound fluid solution.
   4. Stir the wound fluid solution containing the dialysis bag at 200-250 rpm.
   5. Collect the supernatant solution and replace it with an equal quantity of fresh buffer solution at definite time intervals.
   6. Determine the percentage of DOX release from the scaffolds in the supernatant solution using a UV-visible spectrometer at 240 nm.

4. In vitro anti-bacterial studies

1. Determine the minimum inhibitory concentration (MIC) of the CL and DOX-CL scaffolds against the S. aureus, S. epidermis, E. coli, P. aeruginosa using the micro-broth dilution method.
2. Prepare the bacterial cultures using Mueller-Hinton broth at a ratio of 1:1000 to obtain 0.5 McFarland turbidity.
3. Add D-glucose (800 mg/dL) to the bacterial cultures for hyperglycation ^19,20.
4. Mince and solubilize the CL and DOX-CL in DMSO (negative control).
5. Serially dilute the hyperglycated bacterial suspension (100 µL) and test samples (100 µL of scaffolds solution) in 96 well plate.
6. Incubate the plate at 37 °C for 20-24 h.
7. Record the absorbance at a wavelength of 600 nm ²¹.

5. In vitro biocompatibility studies

1. Evaluate the biocompatibility of the prepared scaffolds using MTT [(3-(4, 5 dimethyl thiazole-2 yl) -2, 5-diphenyl tetrazolium bromide)] assay.
2. Sterilize the scaffolds of standard dimension and place them in 24 well plates.
3. Add 3T3-L1 cells to the 24 well plate and incubated for 72 h.

6. In vivo animal studies

1. Induction of DM and excision wound
   1. Feed the animal with a high-fat diet for two weeks and administer a single dose of streptozotocin (STZ) (50 mg/kg body weight) in citrate buffer solution intraperitoneally to Wistar albino rats (180-200 g) for the induction of type-2 diabetes.
   2. Choose the animals with a constant blood glucose of 250 mg/dL for the study.
   3. Randomize the selected animals for the induction of excision wounds.
   4. Anesthetize the diabetic rats using diethyl ether (5 mL was added to the priorly saturated anesthesia chamber) and confirm using the toe pinch method and mucous membrane color.
   5. Shave the dorsal area (Dorsal thoracic, lumbar region) using an aseptic trimmer and blades (A40).
   6. Sterilize the shaved area with an alcoholic swab.
   7. Excise the skin (2 x 2 cm² and a depth of 1 mm) with an aseptic surgical A40 blade on the shaved area to create an open wound.
   8. Divide the animals into three groups (Group 1- Disease control (Control), Group 2- CL scaffold (Placebo), Group 3- DOX CL scaffold), each group consisting of 6 rats.
   9. Affix the CL and DOX CL scaffolds using surgical tape and cover the control group with sterile gauze for 21 days.
   10. Trace the wound area on a sterile OHP sheet and measure the percentage reduction of the wound using the grid method on days 0, 7, 14, and 21 for all groups.
   11. Calculate the percentage wound reduction using the below formulae.
       

7. Histopathological studies

1. Isolate the healed wound area on days 7, 14, and 21, store in formalin solution (10%).
2. Section the tissues using a microtome to obtain a thickness of 6 µm.
3. Mount the sections on a glass slide and stain using Hematoxylin and eosin ¹⁷.
4. Capture the images under 40x magnification using a digital microscope.

8. Hydroxyproline estimation

1. Isolate the healed wound area on days 0, 7, 14, and 21 for evaluation.
2. Estimate the hydroxyproline content using the procedure described by Reddy G et al., 1996 ²².

9. Elisa test

1. Estimate the MMP-9 levels using the Elisa kit as per the manufacturer's instructions.
2. Isolate the tissue samples from the healed wound area on day 21 and mince using a tissue homogenizer.
3. Centrifuge the obtained homogenate and collect the supernatant.
4. Dilute the supernatant at 100-fold using assay buffer.
5. Scan the plate using a multiple plate reader.

10. Statistical analysis

1. Represent the obtained outcomes as Mean ± SD.
2. Perform the statistical analysis using Graph pad prism v5.01.
3. Attain the statistical significance using One Way Analysis of Variance (ANOVA) and Dunnet's post hoc test.
4. Consider the values with p≤0.05 as significant.

Results

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 ...

Discussion

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...

Materials

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