Introduction: Large burns, greater than 40% TBSA (Total Body Surface Area) are seen in both military and civilian populations [1]. The use of skin substitutes in large burn wounds results in limited success of the procedure due to poor integration and insufficient revascularization of the graft to the wounded area [2],[3]. To address this problem, we have developed a hydrogel using blood plasma and polyethylene glycol (PEG) that will aid in wound vascularization. Being cognizant to develop a clinically usable end-products we have developed a freeze dried PEGylated plasma which can be reconstituted ‘in situ’ forming a hydrogel at the site that will conform to wound margins. With the addition of the human adipose derived stem cells (ASCs) we were able to develop a vascularized hydrogel capable of providing an angiogenic microenvironment that will induce active healing process.
Methods: Platelet free plasma (PFP) was PEGylated (PEG-PFP) by mixing with succinimidyl glutarate PEG followed by incubating for 20 minutes in a 5% CO2 incubator at 37°C [4]. The PEG-PFP liquid mixture was frozen overnight at -80°C and then freeze dried by subjecting them to a series of wet-to-dry cycles in a lyophilizer to obtain freeze dried PEGylated PFP (PEG-FDP). PEG-FDP was reconstituted and simultaneously hydrogelated with human thrombin (12.5U/ml) and incubated for 20 minutes in a 5% CO2 incubator at 37°C. Biophysical characteristics of the PEG-FDP hydrogels were analyzed by rheological and electron microscopic techniques. To prepare ASCs incorporated PEG-FDP, cell suspension (1×105 cells/ml) were soak-loaded first followed by hydrogelating with thrombin. The hydrogels with ASCs were maintained in culture in a 5% CO2 humidified incubator at 37°C for 11 days using MesenPro complete growth media. Phenotypic changes of ASCs within the hydrogels were analyzed using immunofluorescence techniques. PFP and ASCs were derived from human blood and abdominoplasty samples obtained from USAISR burn center under IRB approved protocols H-10-023 and H-11-003, respectively.
Results and Discussion: PEG-PFP following freeze drying process (Figure 1A) were able to easily reconstitute and form a stable hydrogel (Figure 1B) similar to freshly prepared PEG-PFP hydrogels. Scanning electron microscopic analysis showed PEG-FDP hydrogels had highly dense and porous morphology Figure 1C) with no significant damage upon lyophilization process.
ASCs within PEG-FDP hydrogels formed a tubular network-like structure (Figure 2A) and were morphologically similar to cells forming networks within freshly prepared PEG-PFP hydrogels (Figure 2B). The ASCs within the PEG-PDP hydrogels stained for lectin show its tubular structure within the hydrogel (Figure 2C)
Conclusion: In this study we describe a regenerative medicine approach using human blood plasma and ASCs to improve burn wound healing. Our results indicate that lyophilized PEG-FDP can be easily reconstituted as a stable hydrogel and used by clinicians as a ‘point-of-care’ treatment option. In addition we demonstrate that the ASCs delivered via a PEG-FDP hydrogels may aid revascularization of burn wounds and improve engraftment of skin substitutes. Further, the freeze dried PEGylated-PFP have the potential to become a commercially available product for a point of injury treatment option.
Authors would like to thank Dr.Tao You for his technical assistance in performing Scanning Electron Microscopic Experiments
References:
[1] Kauvar DS, et al. Burns 2006;32:853-857.
[2] Supp DM , et al. Clin Dermatol. 2005;23:403-12.
[3] Park KM, et al. Development. 2014;14:2760-9.
[4] Zhang G et al. Tissue Eng. 2006;12(1):9-19.