Acute and Chronic Wound Fluid Inversely Influence Wound Healing in an in-Vitro 3D Wound Model

If a wound progressively heals or the healing process is impaired is basically influenced by the surrounding milieu. This is reflected by the wound fluid. Its specific composition triggers the migration, proliferation and differentiation of dermal and epidermal cells which so far was not sufficiently examined in 2D cell culture models. The influence of the different wound entities was analyzed on a newly implemented three dimensional in-vitro model, which improved the transferability to the in-vivo situation. The influence of pooled wound fluids from patients suffering from acute or chronic wounds were investigated within a time period of 10 days after wound application. Histological and immunohistochemical analyses were performed addressing the impact of AWF and CWF on regeneration, such as cell proliferation, fibroblast activity and cell migration. AWF slightly stimulated fibroblast migration while CWF inhibited their activation and migration. The CXCR4immunopositive population was continuously decreased compared to the control and AWF treatment. The expression of FAP was enhanced under AWF and medium. In keratinocytes CWF massively stimulated cell proliferation initiating on day six after injury. The presence of 10% CWF inhibited fibroblast activation and migration and induced the degradation of the collagen matrix. Keratinocytes were stimulated to proliferate, resulting in healing inhibiting hyperplasia. Transferred to human wounds, no effective wound closure would be achieved because of the de-regulation of pro-proliferative and migration-stimulating factors and a degraded extracellular matrix. This newly implemented 3D study model represents a novel appropriate in-vitro system for studying healing mechanisms and potential therapeutic applications. DOI : COMING SOON Corresponding author: Besser, Manuela, Dr. rer. nat. Dpt. of Translational Wound Research; Centre for Biomedical Education and Research (ZBAF); Witten/Herdecke University; Stockumer Street 10; D-58453 Witten-Annen, E-mail:Manuela.Besser@uni-wh.de, Phone: +49 (0)2302 926 332 Running Title: AWF and CWF affect wound healing


Introduction
Wound healing is a very complex but well-orchestrated process which unfortunately offers a variety of possible opportunities to be disturbed factor-α (TNF-α) and interleukin-1 beta (IL-1β), and matrix metalloproteinases (MMP-2, MMP-9). But no conclusion could be drawn from detection to bioactivity.
Analyses of wound healing processes were often performed on 2D cell culture models or co-cultures [3].
Both did not reflect cellular interaction in a 3 dimensional matrix scaffold. In this study, a 3D wound model comprised of a collagen matrix with integrated fibroblasts enabled more resistance to aggressive cytokines and proteases and mimicked the physiological structure [4,5]. The response of fibroblasts and keratinocytes in a complex wound milieu mimicked by the application of AWF und CWF collected from wound patients was analyzed. The following question was addressed: What is the difference between acute and the chronic wound milieu with regard to the influence on cell proliferation, migration and activation.

Preparation of the Collagen Gel Matrix
The collagen gel disc consisted of 10x DMEM (Biochrom), 1 M NaOH (Merck), dH 2 O (ChemSolute), 7.5% NaHCO 3 (Carl Roth), fibroblast growth medium and rat tail collagen type 1 (Corning; final concentration 2.25 mg/ml). All ingredients were stored on ice before the preparation was started and mixed in the exact order listed above.

Histology and Immunohistochemistry
The 3D wound models were fixed in 4%

Quantitative evaluation
The sections from the 3D-models were wound models independent of the culture condition.
Nevertheless, both cell types were vital and actively migrating in the 3D wound model systems (Fig. 3a).
The number of CXCR4 and FAP-expressing fibroblasts increased in the control medium within the first 2 days after wound apply, reflecting their activation and the induction of migration (Fig. 3b, c). Afterwards, the CWF. The latter was detected for FAP (Fig. 3c).
Regarding the activation of fibroblasts into a migratory phenotype, faint differences were evaluated between AWF supplementation and control medium.

Discussion
The Amongst others, the hyperproliferative state was attributed to a regulation by an autologous mechanism.
It was described, that keratinocyte express IL-1α to induce fibroblasts to express and secrete mitogens for CXCR4-expression with ongoing wound healing time.
The migration was slightly but not significantly increased in contrast to the application of chronic wound fluid. The presence of CWF inhibited the activation as well as the migratory state of fibroblasts ( Fig. 2 a/b; Fig. 3 [22][23][24]. This was impressively supported by the histological characterization after 10 days under CWF challenge in this study (Fig. 1).
Having a closer look on the influence of AWF or CWF on the different dermal cell types, the conclusion could be drawn that fibroblasts react more sensitive or faster to the presence of acute or chronic wound fluid. This observation underlines the findings of Lim et al. [25].
This feature represents the physiological situation, where fibroblasts are one of the first cell types reacting on the environmental cues from inflammatory cells. Like described above, they differ in the composition and density of surface receptors. Both, the positive influence of AWF as well as the toxic features of CWF were reflected predominantly by fibroblasts. The histologic slides of 3D wound models after a 10 days treatment with CWF showed this most impressively (Fig. 1): Keratinocytes survived in a multilayered structure on the destroyed collagen residues while fibroblasts were lacking or apoptotic. Negative effects of CWF were described in 2D cultures [3,26].
In summary, this 3D wound mimicked the direct interaction of fibroblasts and keratinocytes in wounds