FLUORINATED METHACRYLAMIDE CHITOSAN HYDROGEL IMPROVES CELLULAR WOUND HEALING PROCESSESSridhar 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 factorsaffecting it, including age, stress, and diabetes. Diabetes mellitusdecelerates wound healing processes and often leads to non-healingchronic wounds. A key underlying problem in diabetic chronic wounds isthe low availability of oxygen, which leads to stalled wound healing.Current clinical therapies to treat non-healing chronic wounds arehyperbaric oxygen therapy and topical oxygen therapy, but they are noteconomically viable and inconvenient for the patient. Simple solutions arerequired to treat chronic wounds in an economically viable way toovercome the shortcomings of currently available clinical therapies.

Here, we are proposing a chitosan-based hydrogel incorporatingperfluorocarbons (PFCs), which can supply oxygen to chronic wounds,which we call fluorinated methacrylamide chitosan (MACF). MACFhydrogels can be loaded with oxygen and can supply it locally to oxygen-deficient environments. This work presents in vitro studies on human skincells (human dermal fibroblasts and human epidermal keratinocytes) toevaluate the potentially beneficial effects of MACF to wound healing underboth normoxic (21% O2) and hypoxic (1% O2) environments.

Fluorinated methacrylamide chitosan polymer hydrogels were prepared aspreviously reported from our lab [1]. Our intermediate product,methacrylamide chitosan (MAC), was also included in our studies to test ifmethacrylation is providing any benefit in wound healing processes otherthan its primary purpose of enabling crosslinking of the hydrogel. Initiallywe 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 wasperformed 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, cellmetabolism, and total cell number, cells were allowed to proliferate withand without MACF gels for 72 hours; then, PrestoBlue, PicoGreen, andadenosine tri-phosphate (ATP) quantification assays were performed.

RESULTS

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hemisection

MATERIALS and METHODS DISCUSSION

These results demonstrate the benefits of oxygenated MACF hydrogels inimproving cellular functions such as cell migration, total cell number,metabolism and ATP synthesis in hypoxic environments, which is similar to theenvironment 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 andsupply it to necessary places such as oxygen deficient chronic wounds for morethan 48 hours (Fig. 1). Supplying oxygen through MACF gels demonstratedbeneficial results in vitro. It is evident from the results in Figures 2 to 5 thatMACF improved cellular functions involved in wound healing such as cellmigration and cell viability (metabolism) under hypoxic conditions in bothhuman dermal fibroblasts and human epidermal keratinocytes. MACF treatmentimproved total cell number (Fig. 6 A&B) by improving proliferation or replicationin both the cell types under both hypoxia and normoxia. ATP quantification(Fig. 6 C&D ) also revealed that MACF treatments improved cellular ATP levelssignificantly over controls under both normoxia and hypoxia. Previous reportshave shown that under hypoxia ATP levels drop by 60% compared to normoxia[2]. Our MACF treatment partially restored ATP values under hypoxia (Fig. 8C&D). Previous reports have shown that cell proliferation (total cell number) isdecelerated under hypoxic conditions as in chronic wounds, but here MACFimproved cell proliferation partially.

INTRODUCTION RESULTS RESULTS

Figure 4: Effect of oxygenated MACF gel on cell migration. In vitro scratch assay isperformed on confluent monolayer to study cell migration . Cell migration, which isdecelerated under hypoxic conditions, is improved with the application of MACF in bothhuman dermal fibroblasts and human epidermal keratinocytes. Different letters implysignificant difference by one factor ANOVA (p<0.0001).

Figure 5: Effect of oxygenated MACF gel on cellular metabolic activity. Cellular metabolicactivity is evaluated by the application of Presto Blue reagent. Under both hypoxia andnormoxia, application of MACF gel has shown increased metabolic activity in both humandermal fibroblasts and human epidermal keratinocytes. Different letters imply significantdifference by one factor ANOVA (p<0.0001).

Figure 1: Application of oxygenated MACF gel elevated PO2 level in culture mediawith highest value observed at 2 hours after application; these values decreasedthereafter. The PO2 level with MACF is always higher than MAC and no gel controls atall time points. Mean +/- SD with n=4.

Figure 3: Effect of MACF on human epidermal keratinocytes migration. Cell migration isstudied by in vitro scratch assay. At 24 hours, cell migration is 100% completed innormoxia 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 studiedby in vitro scratch assay. At 24 hours, cell migration is 100% completed in normoxiacontrols 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 andATP level respectively. Application of MACF improved cell proliferation and ATP level inboth human skin cells under both normoxia and hypoxia Different letters imply significantdifference by one factor ANOVA (p<0.0001).