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A Mouse Model of Mechanotransduction-driven, Human-like Hypertrophic Scarring

Hypertrophic scarring (HTS) is an abnormal process of wound healing that results in excessive scar tissue formation. Over the past decade, we have ...

University of Arizona

Burns and other traumatic injuries represent a substantial biomedical burden. The current standard of care for deep injuries is autologous split-thickness skin grafting (STSG), which frequently results in contractures, abnormal pigmentation, and loss of biomechanical function. Currently, there are no effective therapies that can prevent fibrosis and contracture after STSG. Here, we have developed a clinically relevant porcine model of STSG and comprehensively characterized porcine cell populations involved in healing with single-cell resolution. We identified an up-regulation of proinflammatory and mechanotransduction signaling pathways in standard STSGs. Blocking mechanotransduction with a small-molecule focal adhesion kinase (FAK) inhibitor promoted healing, reduced contracture, mitigated scar formation, restored collagen architecture, and ultimately improved graft biomechanical properties. Acute mechanotransduction blockade up-regulated myeloid CXCL10-mediated anti-inflammation with decreased CXCL14-mediated myeloid and fibroblast recruitment. At later time points, mechanical signaling shifted fibroblasts toward profibrotic differentiation fates, and disruption of mechanotransduction modulated mesenchymal fibroblast differentiation states to block those responses, instead driving fibroblasts toward proregenerative, adipogenic states similar to unwounded skin. We then confirmed these two diverging fibroblast transcriptional trajectories in human skin, human scar, and a three-dimensional organotypic model of human skin. Together, pharmacological blockade of mechanotransduction markedly improved large animal healing after STSG by promoting both early, anti-inflammatory and late, regenerative transcriptional programs, resulting in healed tissue similar to unwounded skin. FAK inhibition could therefore supplement the current standard of care for traumatic and burn injuries.

Disrupting mechanotransduction decreases fibrosis and contracture in split-thickness skin grafting.

'Smart' bandages based on multimodal wearable devices could enable real-time physiological monitoring and active intervention to promote healing of chronic wounds. However, there has been limited development in incorporation of both sensors and stimulators for the current smart bandage technologies. Additionally, while adhesive electrodes are essential for robust signal transduction, detachment of existing adhesive dressings can lead to secondary damage to delicate wound tissues without switchable adhesion. Here we overcome these issues by developing a flexible bioelectronic system consisting of wirelessly powered, closed-loop sensing and stimulation circuits with skin-interfacing hydrogel electrodes capable of on-demand adhesion and detachment. In mice, we demonstrate that our wound care system can continuously monitor skin impedance and temperature and deliver electrical stimulation in response to the wound environment. Across preclinical wound models, the treatment group healed ~25% more rapidly and with ~50% enhancement in dermal remodeling compared with control. Further, we observed activation of proregenerative genes in monocyte and macrophage cell populations, which may enhance tissue regeneration, neovascularization and dermal recovery.

Wireless, closed-loop, smart bandage with integrated sensors and stimulators for advanced wound care and accelerated healing.

While wound healing in humans occurs primarily through re-epithelization, in rodents it also occurs through contraction of the panniculus carnosus, an underlying muscle layer that humans do not possess. Murine experimental models are by far the most convenient and inexpensive research model to study wound healing, as they offer great variability in genetic alterations and disease models. To overcome the obstacle of contraction biasing wound healing kinetics, our group invented the splinted excisional wound model. While other rodent wound healing models have been used in the past, the splinted excisional wound model has persisted as the most used model in the field of wound healing. Here, we present a detailed protocol of updated and refined techniques necessary to utilize this model, generate results with high validity, and accurately analyze the collected data. This model is simple to conduct and provides an easy, standardizable, and replicable model of human-like wound healing.

Protocol for the Splinted, Human-like Excisional Wound Model in Mice.

According to the American Diabetes Association (ADA), 9-12 million patients suffer from chronic ulceration each year, costing the healthcare system over USD $25 billion annually. There is a significant unmet need for new and efficacious therapies to accelerate closure of non-healing wounds. Nitric Oxide (NO) levels typically increase rapidly after skin injury in the inflammatory phase and gradually diminish as wound healing progresses. The effect of increased NO concentration on promoting re-epithelization and wound closure has yet to be described in the context of diabetic wound healing.

Nitric oxide-releasing gel accelerates healing in a diabetic murine splinted excisional wound model.

Chronic wounds impose a significant healthcare burden to a broad patient population. Cell-based therapies, while having shown benefits for the treatment of chronic wounds, have not yet achieved widespread adoption into clinical practice. We developed a CRISPR/Cas9 approach to precisely edit murine dendritic cells to enhance their therapeutic potential for healing chronic wounds. Using single-cell RNA sequencing of tolerogenic dendritic cells, we identified N-myc downregulated gene 2 (Ndrg2), which marks a specific population of dendritic cell progenitors, as a promising target for CRISPR knockout. Ndrg2-knockout alters the transcriptomic profile of dendritic cells and preserves an immature cell state with a strong pro-angiogenic and regenerative capacity. We then incorporated our CRISPR-based cell engineering within a therapeutic hydrogel for in vivo cell delivery and developed an effective translational approach for dendritic cell-based immunotherapy that accelerated healing of full-thickness wounds in both non-diabetic and diabetic mouse models. These findings could open the door to future clinical trials using safe gene editing in dendritic cells for treating various types of chronic wounds.

Cas9-mediated knockout of Ndrg2 enhances the regenerative potential of dendritic cells for wound healing.

Small animals do not replicate the severity of the human foreign-body response (FBR) to implants. Here we show that the FBR can be driven by forces generated at the implant surface that, owing to allometric scaling, increase exponentially with body size. We found that the human FBR is mediated by immune-cell-specific RAC2 mechanotransduction signalling, independently of the chemistry and mechanical properties of the implant, and that a pathological FBR that is human-like at the molecular, cellular and tissue levels can be induced in mice via the application of human-tissue-scale forces through a vibrating silicone implant. FBRs to such elevated extrinsic forces in the mice were also mediated by the activation of Rac2 signalling in a subpopulation of mechanoresponsive myeloid cells, which could be substantially reduced via the pharmacological or genetic inhibition of Rac2. Our findings provide an explanation for the stark differences in FBRs observed in small animals and humans, and have implications for the design and safety of implantable devices.

Allometrically scaling tissue forces drive pathological foreign-body responses to implants via Rac2-activated myeloid cells.

 Given the significant economic, health care, and personal burden of acute and chronic wounds, we investigated the dose dependent wound healing mechanisms of two  derived compounds: avenanthramide (AVN) and β-Glucan.  We utilized a splinted excisional wound model that mimics human-like wound healing and performed subcutaneous AVN and β-Glucan injections in 15-week-old C57BL/6 mice. Histologic and immunohistochemical analysis was performed on the explanted scar tissue to assess changes in collagen architecture and cellular responses.  AVN and β-Glucan treatment provided therapeutic benefits at a 1% dose by weight in a phosphate-buffered saline vehicle, including accelerated healing time, beneficial cellular recruitment, and improved tissue architecture of healed scars. One percent AVN treatment promoted an extracellular matrix (ECM) architecture similar to unwounded skin, with shorter, more randomly aligned collagen fibers and reduced inflammatory cell presence in the healed tissue. One percent β-Glucan treatment promoted a tissue architecture characterized by long, thick bundles of collagen with increased blood vessel density.  AVN and β-Glucan have previously shown promise in promoting wound healing, although the therapeutic efficacies and mechanisms of these bioactive compounds remain incompletely understood. Furthermore, the healed ECM architecture of these wounds has not been characterized.  AVN and β-Glucan accelerated wound closure compared to controls through distinct mechanisms. AVN-treated scars displayed a more regenerative tissue architecture with reduced inflammatory cell recruitment, while β-Glucan demonstrated increased angiogenesis with more highly aligned tissue architecture more indicative of fibrosis. A deeper understanding of the mechanisms driving healing in these two naturally derived therapeutics will be important for translation to human use.

Avenanthramide and β-Glucan Therapeutics Accelerate Wound Healing Via Distinct and Nonoverlapping Mechanisms.

The FOXC2 transcription factor has been tied to a wide range of disease states, serving as a promising prognostic biomarker associated with aggressive basal-like human breast cancers (increased cancer invasion and metastasis). Dysregulation of FOXC2 expression has also been found to promote defects in lymphatic remodeling and hyperplastic lymphedema-distichiasis (LD). Since chronic lymphedema is a forerunner of several malignancies and cancers have been known to arise from poorly healing chronic wounds (e.g., Marjolin ulcers), we examined the effect of Foxc2 dysfunction on skin wound healing.

Hudson C. Kussie

Department of Surgery

University of Arizona

A Mouse Model of Mechanotransduction-driven, Human-like Hypertrophic Scarring

Hudson C. Kussie

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Surgery

University of Arizona

A Mouse Model of Mechanotransduction-driven, Human-like Hypertrophic Scarring

Department of Surgery