Evaluating the Biocompatibility of an Injectable Wound Matrix in a Murine Model

(1) Background: Developing a high-quality, injectable biomaterial that is labor-saving, cost-efficient, and patient-ready is highly desirable. Our research group has previously developed a collagen-based injectable scaffold for the treatment of a variety of wounds including wounds with deep and irregular beds. Here, we investigated the biocompatibility of our liquid scaffold in mice and compared the results to a commercially available injectable granular collagen-based product. (2) Methods: Scaffolds were applied in sub-dermal pockets on the dorsum of mice. To examine the interaction between the scaffolds and the host tissue, samples were harvested after 1 and 2 weeks and stained for collagen content using Masson’s Trichrome staining. Immunofluorescence staining and quantification were performed to assess the type and number of cells infiltrating each scaffold. (3) Results: Histological evaluation after 1 and 2 weeks demonstrated early and efficient integration of our liquid scaffold with no evident adverse foreign body reaction. This rapid incorporation was accompanied by significant cellular infiltration of stromal and immune cells into the scaffold when compared to the commercial product (p < 0.01) and the control group (p < 0.05). Contrarily, the commercial scaffold induced a foreign body reaction as it was surrounded by a capsule-like, dense cellular layer during the 2-week period, resulting in delayed integration and hampered cellular infiltration. (4) Conclusion: Results obtained from this study demonstrate the potential use of our liquid scaffold as an advanced injectable wound matrix for the management of skin wounds with complex geometries.Medicine, Faculty ofNon UBCSurgery, Department ofReviewedFacultyResearcherOthe

An Evaluation of the Treatment of Full-Thickness Wounds Using Adipose Micro-Fragments within a Liquid Dermal Scaffold

In full-thickness wounds, inflammation, lack of matrix deposition, and paucity of progenitor cells delay healing. As commercially available solid (sheet) scaffolds are unable to conform to wounds of varying shapes and sizes, we previously generated a nutritious, injectable, liquid skin substitute that can conform to wound topography. In combination with adipose micro-fragments as a viable source of progenitor cells, a composite, in situ forming skin substitute was tested for the treatment of silicon ring splinted full-thickness wounds in rats. The in vitro survivability and migratory capacity of adipocytes derived from rat micro-fragmented fat cultured in our scaffold was examined with a Live/Dead assay, showing viability and migration after 7 and 14 days. In vivo, the efficacy of our scaffold alone (LDS) or with adipose micro-fragments (LDS+A) was compared to a standard dressing protocol (NT). LDS and LDS+A showed ameliorated wound healing, including complete epithelialization and less immune cell infiltration, compared to the NT control. Our findings demonstrate that a 3D liquid skin scaffold is a rich environment for adipocyte viability and migration, and that the addition of adipose micro-fragments to this scaffold can be used as a rich source of cells for treating full-thickness wounds

Anti-Scarring Properties of Different Tryptophan Derivatives

<div><p>Hypertrophic scars are associated with prolonged extracellular matrix (ECM) production, aberrant ECM degradation and high tissue cellularity. Routinely used antifibrotic strategies aim to reduce ECM deposition and enhance matrix remodeling. Our previous study investigating the antifibrotic effects of indoleamine2, 3 dioxygenase (IDO) led to the identification of kynurenine (Kyn) as an antiscarring agent. A topical antifibrogenic therapy using Kyn is very attractive; however, it is well established that Kyn passes the blood brain barrier (BBB) which causes complications including excitatory neuronal death. Here we investigated the antiscarring properties of kynurenic acid (KynA), a downstream end product of Kyn that is unlikely to pass the BBB, as an effective and safe replacement for Kyn. Our results indicated that while not having any adverse effect on dermal cell viability, KynA significantly increases the expression of matrix metalloproteinases (MMP1 and MMP3) and suppresses the production of type-I collagen and fibronectin by fibroblasts. Topical application of cream containing KynA in fibrotic rabbit ear significantly decreased scar elevation index (1.13±0.13 vs. 1.61±0.12) and tissue cellularity (221.38±21.7 vs. 314.56±8.66 cells/hpf) in KynA treated wounds compared to controls. KynA treated wounds exhibited lower levels of collagen deposition which is accompanied with a significant decrease in type-I collagen and fibronectin expression, as well as an increase in MMP1 expression compared to untreated wounds or wounds treated with cream only. The results of this study provided evidence for the first time that KynA is promising candidate antifibrogenic agent to improve healing outcome in patients at risk of hypertrophic scarring.</p></div

Stimulatory effect of kynurenines on MMP1 expression.

<p><b>A</b>: Dermal fibroblasts were treated with increasing doses (6.25, 12.5, 25, 50, 100, and 150 μg/ml) of KynA, Kyn, L-Kyn or D-Kyn. Following 24 hours of treatment cells were collected, and MMP1 expression was determined by Q-PCR after RNA extraction and cDNA synthesis. <b>B</b>: Evaluation of MMP1 expression at the protein level by Western blotting after 48 hours of treatment. <b>C</b>: The Mean±SEM ratio of MMP1 to β-actin density at the protein level. β-actin and GAPDH were used as loading controls for western blotting and Q-PCR, respectively.</p

Inhibition of type-I collagen and fibronectin expression in dermal fibroblasts by kynurenines.

<p>Type-I Collagen and fibronectin expression at the mRNA and protein level in cultured fibroblasts treated with increasing concentrations (6.25, 12.5, 25, 50, 100, and 150 μg/ml) of KynA, Kyn, L-Kyn or D-Kyn. <b>A & B</b>: Relative type-I collagen and fibronectin mRNA expression in treated fibroblasts, respectively. GAPDH was used as the reference gene. <b>C</b>: Evaluation of type-I collagen and fibronectin expression at the protein level using Western blotting. <b>D & E</b>: The Mean±SEM ratio of type-I collagen and fibronectin density to β-actin at the protein level, respectively. β-actin was used as protein loading control.</p

Stimulatory effect of Kynurenines on MMP activity and Kynurenines lasting effect on MMP1 expression.

<p><b>A</b>: The effect of kynurenines of MMP activity. To determine MMP activity in the fibroblasts conditioned medium, cells were treated with 100 μg/ml of KynA, Kyn, L-Kyn or D-Kyn for 48 hours and MMP activity was evaluated using SensoLyte Plus 520 generic MMP Assay Kit (** P-value<0.001, n = 4). <b>B</b>: Kynurenines lasting effect on the MMP1 expression. To determine the lasting effect of kynurenines on MMP1 expression, fibroblasts were treated with KynA or Kyn (100 μg/ml) for 48 hours. The medium was replaced and cells were harvested immediately, 12, 24, and 48 hours after treatment removal. The MMP1 expression in dermal fibroblasts was evaluated using western blotting. <b>C</b>: MMP1/β-actin expression ratio was calculated in treated fibroblasts. Data is mean±SEM of 4 independent experiments (* P-value<0.05 and ** P-value<0.01, n = 4).</p

Effect of kynurenines on fibroblast and keratinocyte proliferation rate and viability.

<p><b>A & B</b>: To determine the effect of different kynurenines on dermal cell proliferation rate, fibroblast and keratinocytes were treated with KynA or Kyn (100 μg/ml). Cells were harvested and total cell number was counted after 36, 72 and 108 hours of incubation (* P-value<0.05 and ** P-value<0.01, n = 6). <b>C</b>: Determination of cellular viability by FACs analysis using live/dead, viability/cytotoxicity assay kit. Fibroblasts and keratinocytes were either cultured in DMEM+2% FBS or DMEM+2% FBS supplemented with increasing concentrations of KynA or Kyn (50 and 150 μg/ml). The viability of cells was determined by FACS analysis following 3 days of incubation.</p

Effect of kynurenines on fibroblast and keratinocyte migration.

<p><b>A & B</b>: Images of human fibroblasts and keratinocytes taken immediately, 12 and 24 h after addition of KynA or Kyn (100 μg/ml) in an <i>in vitro</i> wound scratch assay, respectively. <b>C</b>: Reduction of fibroblast migration in the presence of KynA after 12 and 24 hours of treatment. <b>D</b>: Enhancement of keratinocyte migration in the presence of KynA and Kyn. Cell migration was evaluated by manually counting the total number of cells migrated from the edges of the wound into the denuded area (* P-value<0.05 and ** P-value<0.01, n = 4).</p

Effect of Kyn and KynA topical application on collagen deposition, tissue cellularity and ECM expression.

<p>A: Evaluation of collagen deposition in tissue samples using Masson's Trichrome staining at day 35 post-wounding. In this staining collagen fibers are stained blue, keratin and muscle fibers are stained red, and cell cytoplasm and nuclei are stained light pink and dark brown, respectively. B: Quantification and statistical analysis of tissue cellularity. Q-PCR analysis of relative MMP1 (C), type-I collagen (D) and fibronectin (E) mRNA expression in tissue samples (* P-value<0.05 and ** P-value<0.01, n = 4).</p

Stimulatory effect of kynurenines on MMP3 secretion by fibroblasts.

<p><b>A</b>: Evaluation of MMP3 mRNA expression in fibroblasts treated with increasing doses (6.25, 12.5, 25, 50, 100, and 150 μg/ml) of KynA, Kyn, L-Kyn or D-Kyn following 24 hours of treatment. GAPDH was used as loading control for Q-PCR. <b>B</b>: Evaluation of MMP3 presence in the fibroblast conditioned medium using Western blotting after 48 hours of treatment. <b>C</b>: The Mean±SEM ratio of MMP3 density at the protein level.</p