1. FLUORINATED METHACRYLAMIDE CHITOSAN HYDROGEL IMPROVES CELLULAR WOUND HEALING PROCESSES
Sridhar Akula , Nic D. Leipzig
Department of Chemical and Biomolecular engineering, The University of Akron, Akron, OH, USA
Email: sa170@uakron.edu, nl21@uakron.edu
A B C D E F G H
200µm
CONCLUSIONS
Wound healing is a complex process with many local and systematic factors
affecting it, including age, stress, and diabetes. Diabetes mellitus
decelerates wound healing processes and often leads to non-healing
chronic wounds. A key underlying problem in diabetic chronic wounds is
the low availability of oxygen, which leads to stalled wound healing.
Current clinical therapies to treat non-healing chronic wounds are
hyperbaric oxygen therapy and topical oxygen therapy, but they are not
economically viable and inconvenient for the patient. Simple solutions are
required to treat chronic wounds in an economically viable way to
overcome the shortcomings of currently available clinical therapies.
Here, we are proposing a chitosan-based hydrogel incorporating
perfluorocarbons (PFCs), which can supply oxygen to chronic wounds,
which we call fluorinated methacrylamide chitosan (MACF). MACF
hydrogels can be loaded with oxygen and can supply it locally to oxygen-
deficient environments. This work presents in vitro studies on human skin
cells (human dermal fibroblasts and human epidermal keratinocytes) to
evaluate the potentially beneficial effects of MACF to wound healing under
both normoxic (21% O2) and hypoxic (1% O2) environments.
Fluorinated methacrylamide chitosan polymer hydrogels were prepared as
previously reported from our lab [1]. Our intermediate product,
methacrylamide chitosan (MAC), was also included in our studies to test if
methacrylation is providing any benefit in wound healing processes other
than its primary purpose of enabling crosslinking of the hydrogel. Initially
we studied the oxygen levels in the culture media with MACF gel on it.
Next, to study the effect of MACF on cell migration, a scratch assay was
performed on confluent monolayers of human cells grown in 24 well-
plates. The resulting percentage of scratch closure was compared controls
(no gel) and MACF gel treatments at 24 hours under both normoxia (N)
and hypoxia (H). To evaluate the effects of MACF on cell morphology, cell
metabolism, and total cell number, cells were allowed to proliferate with
and without MACF gels for 72 hours; then, PrestoBlue, PicoGreen, and
adenosine tri-phosphate (ATP) quantification assays were performed.
RESULTS
2 mm
hemisection
MATERIALS and METHODS DISCUSSION
These results demonstrate the benefits of oxygenated MACF hydrogels in
improving cellular functions such as cell migration, total cell number,
metabolism and ATP synthesis in hypoxic environments, which is similar to the
environment found around chronic wounds.
We would like to acknowledge funding from the NIH (R15GM104851).
1. Wijekoon A., et al., Acta Biomaterialia, (9) 2013, 5653-64.
2. Baracca A., et al., Int J Biochem Cell Biol, 45 (2013), 135.
REFERENCES
ACKNOWLEDGEMENT
This study once again demonstrated that MACF can be loaded with oxygen and
supply it to necessary places such as oxygen deficient chronic wounds for more
than 48 hours (Fig. 1). Supplying oxygen through MACF gels demonstrated
beneficial results in vitro. It is evident from the results in Figures 2 to 5 that
MACF improved cellular functions involved in wound healing such as cell
migration and cell viability (metabolism) under hypoxic conditions in both
human dermal fibroblasts and human epidermal keratinocytes. MACF treatment
improved total cell number (Fig. 6 A&B) by improving proliferation or replication
in both the cell types under both hypoxia and normoxia. ATP quantification
(Fig. 6 C&D ) also revealed that MACF treatments improved cellular ATP levels
significantly over controls under both normoxia and hypoxia. Previous reports
have shown that under hypoxia ATP levels drop by 60% compared to normoxia
[2]. Our MACF treatment partially restored ATP values under hypoxia (Fig. 8
C&D). Previous reports have shown that cell proliferation (total cell number) is
decelerated under hypoxic conditions as in chronic wounds, but here MACF
improved cell proliferation partially.
INTRODUCTION RESULTSRESULTS
Figure 4: Effect of oxygenated MACF gel on cell migration. In vitro scratch assay is
performed on confluent monolayer to study cell migration . Cell migration, which is
decelerated under hypoxic conditions, is improved with the application of MACF in both
human dermal fibroblasts and human epidermal keratinocytes. Different letters imply
significant difference by one factor ANOVA (p<0.0001).
Figure 5: Effect of oxygenated MACF gel on cellular metabolic activity. Cellular metabolic
activity is evaluated by the application of Presto Blue reagent. Under both hypoxia and
normoxia, application of MACF gel has shown increased metabolic activity in both human
dermal fibroblasts and human epidermal keratinocytes. Different letters imply significant
difference by one factor ANOVA (p<0.0001).
Figure 1: Application of oxygenated MACF gel elevated PO2 level in culture media
with highest value observed at 2 hours after application; these values decreased
thereafter. The PO2 level with MACF is always higher than MAC and no gel controls at
all time points. Mean +/- SD with n=4.
Figure 3: Effect of MACF on human epidermal keratinocytes migration. Cell migration is
studied by in vitro scratch assay. At 24 hours, cell migration is 100% completed in
normoxia controls whereas in hypoxia controls it is not completed. Scale bars are 200 µm.
Figure 2: Effect of MACF on human dermal fibroblasts migration. Cell migration is studied
by in vitro scratch assay. At 24 hours, cell migration is 100% completed in normoxia
controls whereas in hypoxia controls it is not completed. Scale bars are 200 µm.
Figure 6: Effect of oxygenated MACF gel on total cell number and total ATP content.
PicoGreen assay and ATP determination kit are used to evaluate the total cell number and
ATP level respectively. Application of MACF improved cell proliferation and ATP level in
both human skin cells under both normoxia and hypoxia Different letters imply significant
difference by one factor ANOVA (p<0.0001).