(J-411) Development of Chitosan/PVA Hetero-Composite Hydrogel with Antibacterial, Anti-inflammatory, Oxygen Supply, and Enhanced Cell Growth Functionalities for Diabetic Wound Healing

Introduction:: Diabetic foot ulcer (DFU) remain one of the most difficult-to-treat complications of diabetes and may seriously threaten the life of patients since it frequently results in limb loss due to amputation, suggesting that an effective therapeutic strategy is still urgently needed nowadays. The main principles for DFU treatment are wound debridement, pressure offloading, revascularization and infection management. Hydrogels are commonly proposed as a favorable tool for wound repair since they are able to provide further benefits, including a moist environment, low adhesion, and thermal insulation, to increase the probability of wound healing compared with other dressings. However, coverage may form another barrier to oxygen delivery over the exudates on the wound bed, while oxygen plays a crucial role in many biochemical and cellular processes related to wound healing, such as infection control, construction of the extracellular matrix, and collagen formation/remodeling. In this study, we sought to design, fabricate and explore a new type of chitosan/PVA-based heterogeneous composite hydrogel containing perfluorocarbon (PFC) emulsions (PEs), EGF-loaded nanoparticles (ENPs), and the antimicrobials polyhexamethylene biguanide (PHMB) named P_EE_NPPCH for diabetic wound treatment, in which separate encapsulation of EGF and PFC may prevent their interactions with PHMB and/or with each other before they are released to the wound site. Furthermore, P_EE_NPPCH may exert additional anti-inflammatory effects due to the anti-inflammatory characteristics of chitosan reported previously. With capabilities of oxygen delivery, antimicrobial activity, anti-inflammation, and promotion of cell growth, we anticipate that the developed P_EE_NPPCH may be able to provide improved efficacy in diabetic wound repair.

Materials and Methods:: The P_EE_NPPCH was manufactured by incorporating PEs, ENPs, and PHMB into chitosan/PVA matrix using a modified freeze–thaw cycling method, in which the PEs were fabricated by homogenizing perfluorooctyl bromide (PFOB) and Pluronic F68, and ENPs were prepared by encapsulating EGF into chitosan/sodium tripolyphosphate matrix through emulsification approach. The functionalities of antibacterial, oxygen supply capability, anti-inflammation, and promoted cell growth were assessed by colony assay, resazurin assay, detection of cell IL-8 expression, and measurement of growth rate of human keratinocytes (KERTr cells), respectively. The efficacy of the P_EE_NPPCH on wound healing in vivo was directly evaluated using the diabetic Sprague–Dawley rats as the experimental model. The animal procedures were all accordance with the guidelines approved by the Cathay General Hospital (Taiwan ROC, Approval number: IACUC 110-001). Diabetes mellitus was induced in rats by administering a single intravenous injection of 65 mg/kg streptozotocin (STZ), and rats with blood glucose levels > 300 mg/dL on the 21^st day were considered diabetic. After anaesthetization via isoflurane inhalation, two identical circular full-thickness wounds with d = 0.8 cm were created on the dorsal skin by a punch. One of the two wounds on each rat was covered with sterilized gauze, while the other wound was treated with the commercial dressing HeraDerm, blank chitosan/PVA hydrogel (CH), E_NPPCH, P_EPCH, or P_EE_NPPCH. Dressings were changed every 72 h throughout the experiment. The degree of wound closure (DWC) and levels of reepithelization, deposition of collagen, and integrity of newly formed tissues were comprehensively evaluated in this study.


Results, Conclusions, and Discussions:: Fig. 1A and Fig. 1B show SEM images of ENPs and PEs, respectively. The P_EE_NPPCH is a moist and semitransparent membrane (Fig. 1C) with a porous morphology on both surface and internal structures as illustrated in Fig. 1(D & E). Moreover, it can be seen that a number of nanoparticles (ENPs and PEs) were adhered to the fibrous and/or entrapped inside the hydrogel matrix (Fig. IF), illustrating the heterogeneous construction of the P_EE_NPPCH. The oxygen delivery capability of the P_EE_NPPCH was qualitatively detected using resazurin and the results show that the P_EE_NPPCH containing PFOB is helpful for oxygen delivery/supply (Fig. 2A). The results of colony assay shown in Fig. 2(B-D) indicate that the P_EE_NPPCH may provide significant antibacterial efficacy when the PHMB dosage is ³ 500 ppm. In addition, the P_EE_NPPCH containing ³ 0.6 mg/mL nanoparticle-encapsulated EGF was able to facilitate keratinocyte proliferation (Fig. 3A & 3B)). Based on the IL-8 expression analysis (Fig. 3C), the P_EE_NPPCH was able to reduce the inflammatory response caused by S. aureus and we reason that it was attributed to the chitosan released from the P_EE_NPPCH. For the in vivo study as presented in Fig. 4A, the wound treated with P_EE_NPPCH quickly healed and displayed the smallest visible size among all groups after 15 days (Fig. 4B). Based on the quantitative analysis of the wound area (Fig. 4C), the wound with P_EE_NPPCH showed the greatest DWC of 35% at Day 3 and reached 95% DWC at Day 15. In addition, the histological results show that the P_EE_NPPCH may provide a relatively complete/intact epithelial layer (Fig. 5I) and milder inflammatory response (Fig. 5II) compared to all the other treatments. Furthermore, most of the regenerating collagen in the group with P_EE_NPPCH were aligned in a more horizontal manner and showed a smaller alignment angle (Fig. 5III), demonstrating that they were in an enhanced maturation level which is strongly related to the functionality and integrity of the recovered tissues. Given the aforementioned effectiveness together with known merits of hydrogel, we anticipate that the developed P_EE_NPPCH is highly potential for use in clinical diabetic/chronic wound treatment.

Acknowledgements (Optional): : This work was financially supported by National Science and Technology Council, Taiwan R.O.C. (NSTC 111-2221-E-008 -016 -MY2, YH. Lee)


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