Cellularized Bilayer Pullulan-Gelatin Hydrogel for Skin Regeneration

Abstract

Skin substitutes significantly reduce the morbidity and mortality of patients with burn injuries and chronic wounds. However, current skin substitutes have disadvantages related to high costs and inadequate skin regeneration due to highly inflammatory wounds. Thus, new skin substitutes are needed. By combining two polymers, pullulan, an inexpensive polysaccharide with antioxidant properties, and gelatin, a derivative of collagen with high water absorbency, we created a novel inexpensive hydrogel-named PG-1 for ''pullulan-gelatin first generation hydrogel''-suitable for skin substitutes. After incorporating human fibroblasts and keratinocytes onto PG-1 using centrifugation over 5 days, we created a cellularized bilayer skin substitute. Cellularized PG-1 was compared to acellular PG-1 and no hydrogel (control) in vivo in a mouse excisional skin biopsy model using newly developed dome inserts to house the skin substitutes and prevent mouse skin contraction during wound healing. PG-1 had an average pore size of 61.69 mm with an ideal elastic modulus, swelling behavior, and biodegradability for use as a hydrogel for skin substitutes. Excellent skin cell viability, proliferation, differentiation, and morphology were visualized through live/dead assays, 5-bromo-2¢-deoxyuridine proliferation assays, and confocal microscopy. Trichrome and immunohistochemical staining of excisional wounds treated with the cellularized skin substitute revealed thicker newly formed skin with a higher proportion of actively proliferating cells and incorporation of human cells compared to acellular PG-1 or control. Excisional wounds treated with acellular or cellularized hydrogels showed significantly less macrophage infiltration and increased angiogenesis 14 days post skin biopsy compared to control. These results show that PG-1 has ideal mechanical characteristics and allows ideal cellular characteristics. In vivo evidence suggests that cellularized PG-1 promotes skin regeneration and may help promote wound healing in highly inflammatory wounds, such as burns and chronic wounds.

Methodologies in creating skin substitutes

“…It is a polysaccharide polymer with antioxidant and anti-inflammatory properties making it an attractive polymer to prevent infection and graft rejection[46,78]. Pullulan is also relatively inexpensive when compared to other natural polymers and may be further considered for skin substitutes as the pressure for cheaper medical treatments rises.…”

“…Pullulan is also relatively inexpensive when compared to other natural polymers and may be further considered for skin substitutes as the pressure for cheaper medical treatments rises. Currently, pullulan has only been incorporated into collagen and, most recently, gelatin scaffolds for skin regeneration studies, but these studies have all shown promising preliminary results[44–46,78]. More investigation is needed in the effects of this polymer when incorporated into skin substitutes.…”

Biomaterials for Skin Substitutes

“…Pullulan is a neutral, linear, antioxidant, biodegradable, and nontoxic polysaccharide produced by the fungus Aureobasidium pullulans . Pullulan can be further crosslinked and functionalized through its hydroxyl functional groups.…”

Examining the contribution of surrounding intact skin during cutaneous healing

“…However, there are still limitations to current skin substitutes due to prolonged inflammatory phases and inadequate regeneration in larger wounds (Bello et al 2001;Nyame et al 2014;Shahrokhi et al 2014). Furthermore, although skin substitutes are more available than skin grafts, they are costly and can be more expensive than cadaveric allografts (Jones et al 2002;Debels et al 2015;Nicholas et al 2016;Jeschke et al 2017;Hakimi et al 2018). Thus, novel skin substitutes are being developed and tested in order to improve the outcomes of healing while maintaining lower costs.…”