Modulation of the keratinocyte-fibroblast paracrine relationship with gelatin-based semi-interpenetrating networks containing bioactive factors for wound repair

Abstract

Gelatin-based semi-interpenetrating networks (sIPNs) containing soluble and covalently-linked bioactive factors have been shown to aid in wound healing; however, the biological responses elicited by the introduction of sIPN biomaterials remain unclear. In the current study, modulation of the re-epithelialization phase of wound healing by sIPNs grafted with PEGylated fibronectin-derived peptides and utilized as platforms for the delivery of exogenous keratinocyte growth factor (KGF) was evaluated. Following wounding, keratinocyte migration, proliferation and protein secretion is largely controlled by diffusible factors, such as KGF, released by the underlying fibroblasts. The impact of sIPNs and exogenous KGF upon the latter keratinocyte-fibroblast paracrine relationship and keratinocyte behavior was explored by monitoring keratinocyte adhesion and cytokine (IL-1alpha, IL-1beta, IL-6, KGF, GM-CSF and TGF-alpha) release. Results were generally similar for keratinocyte monoculture and keratinocyte-fibroblast co-culture systems. Although keratinocyte adhesion increased over time for positive control surfaces, adhesion to the sIPNs remained low throughout the course of the study. Release of IL-1alpha and GM-CSF was increased by exogenous KGF. The effects were more noticeable on the positive control surfaces relative to the sIPN surfaces. Regulation of the release of TGF-alpha was surface dependent, while IL-6 release was dependent upon surface type, the inclusion of exogenous KGF and the presence of fibroblasts. The findings indicate that during re-epithelialization, sIPNs containing soluble bioactive factors aid in wound healing primarily by serving as conduits for KGF, which induces the release of other key cytokines involved in tissue repair.

Figure 1

Schematic illustration of the keratinocyte–fibroblast autocrine/paracrine relationship. Bold and italicized factors are associated with the release of KGF by fibroblasts. Keratinocyte-derived IL-1α and IL-1β stimulate fibroblasts to express KGF. IL-6, PTHrP and PDGF-BB further regulate KGF expression. KGF, in turn, enhances keratinocyte proliferation and induces release of TGF-α.

Figure 2

Illustration of the semi-interpenetrating network. PEGdA was cross-linked via radical polymerization in the presence of unmodified gelatin, mPEG-gelatin and gelatin conjugated to peptides via a PEG linker to yield sIPNs.

Figure 3

Illustration of the co-culture setup. Neonatal human epidermal keratinocytes (NHEK) were co-cultured with confluent human dermal fibroblasts (HDF) on sIPN and PC surfaces with and without exogenous KGF. The upper (A) and lower (B) chambers of the assembly were assayed for KGF, IL-1α, IL-1β, IL-6, GM-CSF and TGF-α. As controls, keratinocytes and fibroblasts were grown in monoculture on PC and TCPS, respectively.

Figure 4

Preparation of gelatin grafted with PEGylated peptides. PEG was converted to bis-tBuOAc-PEG, which was hydrolyzed to give bis-COOH-PEG. Conjugation of the desired peptide to gelatin through a PEG linker was achieved by N-hydroxysuccinimide activation. In a similar fashion, monomethoxy PEG and gelatin were coupled via the N-hydroxysuccinimide-activated ester.

Figure 5

Release of IL-1α from keratinocytes grown in co-culture with fibroblasts on various surfaces. Supernatant was collected and analyzed from the top (A) and bottom (B) chambers of co-culture wells without KGF at 2 h ( ), with KGF at 2 h ( ), without KGF at 24 h ( ), with KGF at 24 h ( ), without KGF at 96 h ( ), with KGF at 96 h ( ), without KGF at 168 h ( ) and with KGF at 168 h ( ). Results are displayed as cumulative release. The error bars represent mean±SD. Keratinocytes grown on sIPN surfaces showed almost no IL-1α release. At 168 h, keratinocytes grown on PC in co-culture and monoculture in the presence of exogenous KGF had significantly greater IL-1α release compared to keratinocytes grown on sIPN surfaces.

Figure 6

Release of GM-CSF from keratinocytes grown in co-culture with fibroblasts on various surfaces. Supernatant was collected and analyzed from the top (A) and bottom (B) chambers of co-culture wells without KGF at 2 h ( ), with KGF at 2 h ( ), without KGF at 24 h ( ), with KGF at 24 h ( ), without KGF at 96 h ( ), with KGF at 96 h ( ), without KGF at 168 h ( ) and with KGF at 168 h ( ). Results are displayed as cumulative release. The error bars represent mean±SD. Keratinocytes grown on sIPN surfaces showed almost no IL-1α release. Keratinocytes grown on sIPN surfaces showed almost no release of GM-CSF. At 168 h, keratinocytes grown on PC in co-culture with and without exogenous KGF had significantly greater GM-CSF release compared to keratinocytes grown on sIPN surfaces.

Figure 7

Release of TGF-α from keratinocytes grown in co-culture with fibroblasts on various surfaces. Supernatant was collected and analyzed from the top (A) and bottom (B) chambers of co-culture wells without KGF at 2 h ( ), with KGF at 2 h ( ), without KGF at 24 h ( ), with KGF at 24 h ( ), without KGF at 96 h ( ), with KGF at 96 h ( ), without KGF at 168 h ( ) and with KGF at 168 h ( ). Results are displayed as cumulative release. The error bars represent mean±SD. Keratinocytes grown on sIPN surfaces showed almost no release of TGF-α. At 96 and 168 h, keratinocytes grown on PC in co-culture and monoculture in the presence of exogenous KGF had significantly greater TGF-α release compared to keratinocytes grown on sIPN surfaces. At 168 h, keratinocytes grown on PC in monoculture without exogenous KGF also had significantly greater TGF-α release compared to keratinocytes grown on sIPNs. Monoculture release in the absence of exogenous KGF at 168 h was significantly greater than monoculture in the presence of KGF.

Figure 8

Schematic illustration of the method by which surfaces containing exogenous KGF promote wound healing. KGF taken up by keratinocytes promotes the release of IL-1α, which in turn stimulates the release of GM-CSF by both fibroblasts and keratinocytes.