This document summarizes a study that fabricated and evaluated chitosan/gelatin/PVA hydrogels incorporating different concentrations of honey for wound healing applications. The hydrogels were characterized through SEM imaging, mechanical testing, degradation testing, antibacterial evaluation, and cell proliferation assays. An in vivo study on rats found that hydrogels with 10% and 20% honey accelerated wound closure and maintained a well-structured epidermis containing collagen compared to controls. Overall, the addition of honey improved the hydrogels' antibacterial properties and biocompatibility while degrading sufficiently for wound repair.

1. Presented By: Fariah Qaiser
Presented to: Dr. Fakhar-ud-Din
Fabrication and Evaluation of Chitosan/ Gelatin/ PVA
hydrogel incorporating honey for wound healing
applications: An in vitro, in vivo study
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3. Contents
• Rationale of the study
• Materials used
• Methodology
• In-Vivo Wound Healing
• Results & Discussion
• Conclusion
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4. Rationale of the study
• Problem Statement
The commercial product available and the simple hydrogels lack
efficient anti-bacterial activity with a slight delay in wound healing.
• Rationale
The hydrogel incorporating honey shows excellent anti bacterial
properties and cell behavior resulting in the maintenance of a well-
structured layer of epidermis containing mature collagen and
accelerates the rate of wound healing.
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5. Materials
Alcohol (typical average Mw = 146,000–186,000)
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Chitosan (Mol weight. 75-85%
deacetylated)
Nutrient Broth
Honey (Botanical Origin
Chicory)
Acetic Acid PVA
Ethanol
Nutrient Agar
Gelatin (type A, from porcine
skin)

6. Methodology
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7. Preparation of Honey-Chitosan based Hydrogels
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Chitosan dissolved in 3% v/v
acetic acid solution
pH Adjustment
PVA polymer dissolved in
distilled water
5% w/v gelatin solution prepared by
adding gelatin in the distilled water
All prepared solutions were mixed
with a ratio of 2:1:1 (v/v) of
chitosan, PVA, and gelatin solution
under magnetic stirring
Honey at various concentrations
(0% (H-0), 5% (H-5), 10% (H-10),
and 20% (H-20) v/v) added to
polymer solution
Freeze thaw
cycle
Hydrogel

8. Scanning Electron Microscopy (SEM)
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(SEM) Conductive hydrogels ImageJ software application
Mechanical properties
Universal Tensile
Machine
Samples cut into
rectangular shapes
Immersed in water for 2 h
before the test
Scaffolds exposed to
tensile testing at 5 mm/
minute speed until their
breaking point
The young elastic of
hydrogels was
calculated from the
relevant tensile-strain
curve

9. Dynamic Rheological Behavior Measurements
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Anton Paar - Physica Oscillatory Rheometer
Dynamic and flow properties
measurements
All samples were immersed
in water for 2 h
Biodegradation
Initial weights measured (Wo) 24-well plate Incubator
Washing with de-
ionized water
Linear
Viscoelastic
region

10. Anti-bacterial Evaluation
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Inhibition Zone Method
Cell Proliferation and Biocompatibility
MTT Assay
24-well none treated culture
plates
Incubator Elisa plate reader

11. In Vivo Study
• In vivo experiment was done on rat samples and all of them received
care based on the “Guide for the care and use of laboratory animals”
published by the National Institutes of Health (NIH Publication No.
85–23, revised 1985).
• The local committee in the Faculty of Pharmacy, Tehran University,
Tehran, Iran confirmed this experiment based on “Regulations for
using animals in scientific procedures”.
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12. Wound Closure Mechanism
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Histological Analysis
The histopathological characteristics of wound bed skin including Re-epithelialization, inflammation,
angiogenesis, and collagen deposition were investigated.
Statistical Analysis
ANOVA Test
n=6 rats (3 groups) Ketamine
hydrochloride
Injection
Removing back
hair of rats
Full thickness
Injury
H-10 (PVA/chitosan/gelatin hydrogel with 10% v/v honey)
H-20 (PVA/ chitosan/gelatin hydrogel with 20% v/v honey)
Sterile gauze covering the negative control group (NC).
Photographed

13. Results & Discussion
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14. Characterization of Hydrogel Structure
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Fig. 1. SEM images of freeze-dried hydrogels, containing various concentrations of honey in their formulization. H-0 hydrogel
has a microporous structure with a mean pore size of 37 ± 7 µm, while addition of honey reduces the homogenous structure
and increases the mean pore size of hydrogels.

15. Tensile Strength Properties
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Fig. 2. (A) Tensile behavior of the hydrogels (a) H-0, (b) H-5, (c) H-10, and (d) H-20, and (B) elastic modulus of the
hydrogels without and with different concentrations of honey (*P < 0.05 and **P < 0.01).

16. Degradation of scaffolds
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Fig. 4. Degradation behavior of hydrogels in 7 weeks

17. Antibacterial evaluation
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18. Cell proliferation and biocompatibility
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Fig. 5. MTT assay on days 7, 10, and 14 for different honey concentrations in hydrogels
formulations and tissue culture plate (TCP) as the control group (*P < 0.05 and **P < 0.01).

19. Wound closure and Histological Analysis
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Fig. 6. (a) Macroscopic images of wound sites on different days and (b) Mechanism of wound
closure for different groups of treatment during 20 days.

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Fig. 7. Histological analysis of skin tissue repair. Hematoxylin and eosin (H&E) stained samples on
days 12 and 20. On day 20 samples were investigated with Masson’s Trichrome (MT) and PAS
staining

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22. Conclusion
• The chitosan-based hydrogel (H-0) was composed of both natural and synthetic
polymers with a highly porous and sponge-like microstructure similar to the ECM
structure of the skin.
• The addition of honey to the PVA/chitosan/gelatin hydrogels showed no sign of
toxicity.
• Honey-containing hydrogels had higher rate of cell growth in MTT assay.
• The H-10 group showed the maximum rate of biocompatibility.
• Moreover, the inhibition zone of the hydrogels was increased as the concentration of
honey in the hydrogels was increased.
• However, the addition of honey resulted in weaker mechanical properties and faster
degradation, but the samples had the required tensile strength and viscoelastic
properties for the skin tissue and degraded over a sufficient amount of time for the
wound to repair.
• In vivo results showed that the incorporation of honey in the hydrogel matrix in H-10
and H-20 results in the maintenance of a well structured layer of epidermis containing
mature collagen.
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