Kellen Chen

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• Barrera, J. A., Trotsyuk, A. A., Henn, D., Sivaraj, D., Chen, K., Mittal, S., Mermin-Bunnell, A. M., Larson, M. R., Padmanabhan, J., Kinney, B., Nachbar, J., Sacks, J., Terkonda, S. P., Jeffers, L., & Gurtner, G. C. (2023). Blockchain, Information Security, Control, and Integrity: Who Is in Charge?. Plastic and reconstructive surgery, 152(4), 751e-758e.
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  Blockchain technology has attracted substantial interest in recent years, most notably for its effect on global economics through the advent of cryptocurrency. Within the health care domain, blockchain technology has been actively explored as a tool for improving personal health data management, medical device security, and clinical trial management. Despite a strong demand for innovation and cutting-edge technology in plastic surgery, integration of blockchain technologies within plastic surgery is in its infancy. Recent advances and mainstream adoption of blockchain are gaining momentum and have shown significant promise for improving patient care and information management. In this article, the authors explain what defines a blockchain and discuss its history and potential applications in plastic surgery. Existing evidence suggests that blockchain can enable patient-centered data management, improve privacy, and provide additional safeguards against human error. Integration of blockchain technology into clinical practice requires further research and development to demonstrate its safety and efficacy for patients and providers.
• Fischer, K. S., Litmanovich, B., Sivaraj, D., Kussie, H. C., Hahn, W. W., Hostler, A. C., Chen, K., & Gurtner, G. C. (2023). Protocol for the Splinted, Human-like Excisional Wound Model in Mice. Bio-protocol, 13(3), e4606.
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  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.
• Griffin, M. F., Talbott, H. E., Guardino, N. J., Guo, J. L., Spielman, A. F., Chen, K., Parker, J. B., Mascharak, S., Henn, D., Liang, N., King, M., Cotterell, A. C., Bauer-Rowe, K. E., Abbas, D. B., Diaz Deleon, N. M., Sivaraj, D., Fahy, E. J., Downer, M., Akras, D., , Berry, C., et al. (2023). Piezo inhibition prevents rescues scarring by targeting the adipocyte to fibroblast transition. bioRxiv : the preprint server for biology.
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  While past studies have suggested that plasticity exists between dermal fibroblasts and adipocytes, it remains unknown whether fat actively contributes to fibrosis in scarring. We show that adipocytes convert to scar-forming fibroblasts in response to -mediated mechanosensing to drive wound fibrosis. We establish that mechanics alone are sufficient to drive adipocyte-to- fibroblast conversion. By leveraging clonal-lineage-tracing in combination with scRNA-seq, Visium, and CODEX, we define a "mechanically naïve" fibroblast-subpopulation that represents a transcriptionally intermediate state between adipocytes and scar-fibroblasts. Finally, we show that or -inhibition yields regenerative healing by preventing adipocytes' activation to fibroblasts, in both mouse-wounds and a novel human-xenograft-wound model. Importantly, -inhibition induced wound regeneration even in established scars, a finding that suggests a role for adipocyte-to-fibroblast transition in wound remodeling, the least-understood phase of wound healing. Adipocyte-to-fibroblast transition may thus represent a therapeutic target for minimizing fibrosis via -inhibition in organs where fat contributes to fibrosis.
• Henn, D., Trotsyuk, A. A., Barrera, J. A., Sivaraj, D., Chen, K., Mittal, S., Mermin-Bunnell, A. M., Chattopadhyay, A., Larson, M. R., Kinney, B. M., Nachbar, J., TerKonda, S. P., Reddy, S., Jeffers, L., Sacks, J. M., & Gurtner, G. C. (2023). Robotics in Plastic Surgery: It's Here. Plastic and reconstructive surgery, 152(1), 239-249.
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  Although robotic surgery has been routinely established in other surgical disciplines, robotic technologies have been less readily adopted in plastic surgery. Despite a strong demand for innovation and cutting-edge technology in plastic surgery, most reconstructive procedures, including microsurgery, have continued to necessitate an open approach. Recent advances in robotics and artificial intelligence, however, are gaining momentum and have shown significant promise to improve patient care in plastic surgery. These next-generation surgical robots have the potential to enable surgeons to perform complex procedures with greater precision, flexibility, and control than previously possible with conventional techniques. Successful integration of robotic technologies into clinical practice in plastic surgery requires achieving key milestones, including implementing appropriate surgical education and garnering patient trust.
• Henn, D., Zhao, D., Sivaraj, D., Trotsyuk, A., Bonham, C. A., Fischer, K. S., Kehl, T., Fehlmann, T., Greco, A. H., Kussie, H. C., Moortgat Illouz, S. E., Padmanabhan, J., Barrera, J. A., Kneser, U., Lenhof, H. P., Januszyk, M., Levi, B., Keller, A., Longaker, M. T., , Chen, K., et al. (2023). Cas9-mediated knockout of Ndrg2 enhances the regenerative potential of dendritic cells for wound healing. Nature communications, 14(1), 4729.
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  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.
• Padmanabhan, J., Chen, K., Sivaraj, D., Henn, D., Kuehlmann, B. A., Kussie, H. C., Zhao, E. T., Kahn, A., Bonham, C. A., Dohi, T., Beck, T. C., Trotsyuk, A. A., Stern-Buchbinder, Z. A., Than, P. A., Hosseini, H. S., Barrera, J. A., Magbual, N. J., Leeolou, M. C., Fischer, K. S., , Tigchelaar, S. S., et al. (2023). Allometrically scaling tissue forces drive pathological foreign-body responses to implants via Rac2-activated myeloid cells. Nature biomedical engineering, 7(11), 1419-1436.
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  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.
• Sivaraj, D., Noishiki, C., Kosaric, N., Kiwanuka, H., Kussie, H. C., Henn, D., Fischer, K. S., Trotsyuk, A. A., Greco, A. H., Kuehlmann, B. A., Quintero, F., Leeolou, M. C., Granoski, M. B., Hostler, A. C., Hahn, W. W., Januszyk, M., Murad, F., Chen, K., & Gurtner, G. C. (2023). Nitric oxide-releasing gel accelerates healing in a diabetic murine splinted excisional wound model. Frontiers in medicine, 10, 1060758.
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  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.
• Tevlin, R., Griffin, M., Chen, K., Januszyk, M., Guardino, N., Spielman, A., Walters, S., Gold, G. E., Chan, C. K., Gurtner, G. C., Wan, D. C., & Longaker, M. T. (2023). Denervation during mandibular distraction osteogenesis results in impaired bone formation. Scientific reports, 13(1), 2097.
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  Mandibular distraction osteogenesis (DO) is mediated by skeletal stem cells (SSCs) in mice, which enact bone regeneration via neural crest re-activation. As peripheral nerves are essential to progenitor function during development and in response to injury, we questioned if denervation impairs mandibular DO. C57Bl6 mice were divided into two groups: DO with a segmental defect in the inferior alveolar nerve (IAN) at the time of mandibular osteotomy ("DO Den") and DO with IAN intact ("DO Inn"). DO Den demonstrated significantly reduced histological and radiological osteogenesis relative to DO Inn. Denervation preceding DO results in reduced SSC amplification and osteogenic potential in mice. Single cell RNA sequencing analysis revealed that there was a predominance of innervated SSCs in clusters dominated by pathways related to bone formation. A rare human patient specimen was also analyzed and suggested that histological, radiological, and transcriptional alterations seen in mouse DO may be conserved in the setting of denervated human mandible distraction. Fibromodulin (FMOD) transcriptional and protein expression were reduced in denervated relative to innervated mouse and human mandible regenerate. Finally, when exogenous FMOD was added to DO-Den and DO-Inn SSCs undergoing in vitro osteogenic differentiation, the osteogenic potential of DO-Den SSCs was increased in comparison to control untreated DO-Den SSCs, modeling the superior osteogenic potential of DO-Inn SSCs.
• Abbas, D. B., Lavin, C. V., Fahy, E. J., Griffin, M., Guardino, N., King, M., Chen, K., Lorenz, P. H., Gurtner, G. C., Longaker, M. T., Momeni, A., & Wan, D. C. (2022). Standardizing Dimensionless Cutometer Parameters to Determine Elasticity of Human Skin. Advances in wound care, 11(6), 297-310.
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  Skin fibrosis places an enormous burden on patients and society, but disagreement exists over methods to quantify severity of skin scarring. A suction cutometer measures skin elasticity , but it has not been widely adopted because of inconsistency in data produced. We investigated variability of several dimensionless parameters generated by the cutometer to improve their precision and accuracy. Twenty adult human subjects underwent suction cutometer measurement of normal skin (NS) and fibrotic scars (FS). Using Mode 1, each subject underwent five trials with each trial containing four curves. R0/2/5/6/7 and Q1/2/3 data were collected. Analyses were performed on these calculated parameters. R0/2/5/6/7 and Q1/2 parameters from curves 1 to 4 demonstrated significant differences, whereas these same parameters were not significantly different when only using curves 2-4. Individual analysis of all parameters between curve 1 and every subsequent curve was statistically significant for R0, R2, R5, R6, R7, Q1, and Q2. No differences were appreciated for parameter Q3. Comparison between NS and FS were significantly different for parameters R5, Q1, and Q3. Our study is the first demonstration of accurate comparison between NS and FS using the dimensionless parameters of a suction cutometer. Measured parameters from the first curve of each trial were significantly different from subsequent curves for both NS and FS. Precision and reproducibility of data from dimensionless parameters can therefore be improved by removing the first curve. R5, Q1, and Q3 parameters differentiated NS as more elastic than FS.
• Borrelli, M. R., Griffin, M., Chen, K., Deleon Diaz, N. M., Adem, S., Mascharak, S., Shen, A. H., Ngaage, L. M., Lewis, N., Longaker, M. T., Gurtner, G., Wan, D. C., & Lorenz, H. P. (2022). Profibrotic Signaling Pathways and Surface Markers Are Up-Regulated in Fibroblasts of Human Striae Distensae and in a Mouse Model System. Plastic and reconstructive surgery, 150(2), 327-338.
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  Striae distensae are common disfiguring cutaneous lesions but lack effective treatments because of an incomplete understanding of their pathophysiology. Dermal fibroblasts likely play an important role. The authors investigate the cellular-molecular features distinguishing fibroblasts from human striae distensae and normal skin. The authors also develop a mouse model of striae distensae.
• Chen, K., Henn, D., & Gurtner, G. C. (2022). Holy grail of tissue regeneration: Size. BioEssays : news and reviews in molecular, cellular and developmental biology, 44(9), e2200047.
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  Cells and tissue within injured organs undergo a complicated healing process that still remains poorly understood. Interestingly, smaller organisms respond to injury with tissue regeneration and restoration of function, while humans and other large organisms respond to injury by forming dysfunctional, fibrotic scar tissue. Over the past few decades, allometric scaling principles have been well established to show that larger organisms experience exponentially higher tissue forces during movement and locomotion and throughout the organism's lifespan. How these evolutionary adaptations may affect tissue injury has not been thoroughly investigated in humans. We discuss how these adapations may affect healing and demonstrate that blocking the most evolutionary conserved biologic force sensor enables large organisms to heal after injury with true tissue regeneration. Future strategies to disrupt tissue force sensors may unlock the key to regenerating after injury in a wide range of organ systems.
• Chen, K., Henn, D., Januszyk, M., Barrera, J. A., Noishiki, C., Bonham, C. A., Griffin, M., Tevlin, R., Carlomagno, T., Shannon, T., Fehlmann, T., Trotsyuk, A. A., Padmanabhan, J., Sivaraj, D., Perrault, D. P., Zamaleeva, A. I., Mays, C. J., Greco, A. H., Kwon, S. H., , Leeolou, M. C., et al. (2022). Disrupting mechanotransduction decreases fibrosis and contracture in split-thickness skin grafting. Science Translational Medicine, 14(645). doi:10.1126/scitranslmed.abj9152
• Chen, K., Henn, D., Januszyk, M., Barrera, J. A., Noishiki, C., Bonham, C. A., Griffin, M., Tevlin, R., Carlomagno, T., Shannon, T., Fehlmann, T., Trotsyuk, A. A., Padmanabhan, J., Sivaraj, D., Perrault, D. P., Zamaleeva, A. I., Mays, C. J., Greco, A. H., Kwon, S. H., , Leeolou, M. C., et al. (2022). Disrupting mechanotransduction decreases fibrosis and contracture in split-thickness skin grafting. Science translational medicine, 14(645), eabj9152.
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  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.
• Chen, K., Henn, D., Sivaraj, D., Bonham, C. A., Griffin, M., Kussie, H. C., Padmanabhan, J., Trotsyuk, A. A., Wan, D. C., Januszyk, M., Longaker, M. T., & Gurtner, G. C. (2022). Mechanical Strain Drives Myeloid Cell Differentiation Toward Proinflammatory Subpopulations. Advances in wound care, 11(9), 466-478.
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  After injury, humans and other mammals heal by forming fibrotic scar tissue with diminished function, and this healing process involves the dynamic interplay between resident cells within the skin and cells recruited from the circulation. Recent studies have provided mounting evidence that external mechanical forces stimulate intracellular signaling pathways to drive fibrotic processes. While most studies have focused on studying mechanotransduction in fibroblasts, recent data suggest that mechanical stimulation may also shape the behavior of immune cells, referred to as "mechano-immunomodulation." However, the effect of mechanical strain on myeloid cell recruitment and differentiation remains poorly understood and has never been investigated at the single-cell level. In this study, we utilized a three-dimensional (3D) culture system that permits the precise manipulation of mechanical strain applied to cells. We cultured myeloid cells and used single-cell RNA-sequencing to interrogate the effects of strain on myeloid differentiation and transcriptional programming. Our data indicate that myeloid cells are indeed mechanoresponsive, with mechanical stress influencing myeloid differentiation. Mechanical strain also upregulated a cascade of inflammatory chemokines, most notably from the family. Further understanding of how mechanical stress affects myeloid cells in conjunction with other cell types in the complicated, multicellular milieu of wound healing may lead to novel insights and therapies for the treatment of fibrosis.
• Chen, K., Sivaraj, D., Davitt, M. F., Leeolou, M. C., Henn, D., Steele, S. R., Huskins, S. L., Trotsyuk, A. A., Kussie, H. C., Greco, A. H., Padmanabhan, J., Perrault, D. P., Zamaleeva, A. I., Longaker, M. T., & Gurtner, G. C. (2022). Pullulan-Collagen hydrogel wound dressing promotes dermal remodelling and wound healing compared to commercially available collagen dressings. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society, 30(3), 397-408.
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  Biological scaffolds such as hydrogels provide an ideal, physio-mimetic of native extracellular matrix (ECM) that can improve wound healing outcomes after cutaneous injury. While most studies have focused on the benefits of hydrogels in accelerating wound healing, there are minimal data directly comparing different hydrogel material compositions. In this study, we utilized a splinted excisional wound model that recapitulates human-like wound healing in mice and treated wounds with three different collagen hydrogel dressings. We assessed the feasibility of applying each dressing and performed histologic and histopathologic analysis on the explanted scar tissues to assess variations in collagen architecture and alignment, as well as the tissue response. Our data indicate that the material properties of hydrogel dressings can significantly influence healing time, cellular response, and resulting architecture of healed scars. Specifically, our pullulan-collagen hydrogel dressing accelerated wound closure and promoted healed tissue with less dense, more randomly aligned, and shorter collagen fibres. Further understanding of how hydrogel properties affect the healing and resulting scar architecture of wounds may lead to novel insights and further optimization of the material properties of wound dressings.
• Foster, D. S., Januszyk, M., Delitto, D., Yost, K. E., Griffin, M., Guo, J., Guardino, N., Delitto, A. E., Chinta, M., Burcham, A. R., Nguyen, A. T., Bauer-Rowe, K. E., Titan, A. L., Salhotra, A., Jones, R. E., da Silva, O., Lindsay, H. G., Berry, C. E., Chen, K., , Henn, D., et al. (2022). Multiomic analysis reveals conservation of cancer-associated fibroblast phenotypes across species and tissue of origin. Cancer cell, 40(11), 1392-1406.e7.
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  Cancer-associated fibroblasts (CAFs) are integral to the solid tumor microenvironment. CAFs were once thought to be a relatively uniform population of matrix-producing cells, but single-cell RNA sequencing has revealed diverse CAF phenotypes. Here, we further probed CAF heterogeneity with a comprehensive multiomics approach. Using paired, same-cell chromatin accessibility and transcriptome analysis, we provided an integrated analysis of CAF subpopulations over a complex spatial transcriptomic and proteomic landscape to identify three superclusters: steady state-like (SSL), mechanoresponsive (MR), and immunomodulatory (IM) CAFs. These superclusters are recapitulated across multiple tissue types and species. Selective disruption of underlying mechanical force or immune checkpoint inhibition therapy results in shifts in CAF subpopulation distributions and affected tumor growth. As such, the balance among CAF superclusters may have considerable translational implications. Collectively, this research expands our understanding of CAF biology, identifying regulatory pathways in CAF differentiation and elucidating therapeutic targets in a species- and tumor-agnostic manner.
• Griffin, M. F., Fahy, E. J., King, M., Guardino, N., Chen, K., Abbas, D. B., Lavin, C. V., Diaz Deleon, N. M., Lorenz, H. P., Longaker, M. T., & Wan, D. C. (2022). Understanding Scarring in the Oral Mucosa. Advances in wound care, 11(10), 537-547.
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  Skin inevitably heals with the formation of a fibrotic scar. Patients affected by skin scarring suffer from long-term psychological and physical burdens. Since the discovery of fetal scarless skin-wound healing, research has hoped to identify and mimic scarless healing for adult skin. Oral mucosa healing in adults provides the closest example to fetal scarless healing. Injuries to the oral mucosa heal with very minimal scarring. Understanding the mechanisms through which this process occurs may bring us closer to achieving scarless healing in adults. In this review, we summarize the current evidence that illustrates distinct mechanisms involved in oral mucosal healing. We discuss the role of the oral niche in contributing to wound repair. The intrinsic properties of immune cells, fibroblasts, and keratinocytes within the oral mucosa that support regenerative repair are provided. We highlight the contribution of cytokines, growth factors, and chemokine secretion in permitting a scarless mucosal environment. Furthermore, we discuss the role of stem cell-like progenitor populations in the mucosa that may contribute to wound healing. We also provide suggestions for future studies that are needed to achieve scarless healing in adults. Many characteristics of the oral mucosa have been shown to contribute to decreased scarring, but the specific mechanism(s) is unclear. Advancing our understanding of oral healing may yield therapeutic therapies that can be used to overcome dermal scarring.
• Gurtner, G. C., Chen, K., & Henn, D. (2022). Holy grail of tissue regeneration: Size. BioEssays, 44(9), 2200047. doi:10.1002/bies.202200047
• Henn, D., Fischer, K. S., Chen, K., Greco, A. H., Martin, R. A., Sivaraj, D., Trotsyuk, A. A., Mao, H. Q., Reddy, S. K., Kneser, U., Gurtner, G. C., Schmidt, V. J., & Sacks, J. M. (2022). Enrichment of Nanofiber Hydrogel Composite with Fractionated Fat Promotes Regenerative Macrophage Polarization and Vascularization for Soft-Tissue Engineering. Plastic and reconstructive surgery, 149(3), 433e-444e.
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  Fractionated fat has been shown to promote dermal regeneration; however, the use of fat grafting for reconstruction of soft-tissue defects is limited because of volume loss over time. The authors have developed a novel approach for engineering of vascularized soft tissue using an injectable nanofiber hydrogel composite enriched with fractionated fat.
• Januszyk, M., Griffin, M., Mascharak, S., Talbott, H. E., Chen, K., Henn, D., Spielman, A. F., Parker, J. B., Liang, N. E., Cotterell, A., Guardino, N., Foster, D. S., Wagh, D., Coller, J., Gurtner, G. C., Wan, D. C., & Longaker, M. T. (2022). Multiplexed evaluation of mouse wound tissue using oligonucleotide barcoding with single-cell RNA sequencing. STAR protocols, 4(1), 101946.
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  Despite its rapidly increased availability for the study of complex tissue, single-cell RNA sequencing remains prohibitively expensive for large studies. Here, we present a protocol using oligonucleotide barcoding for the tagging and pooling of multiple samples from healing wounds, which are among the most challenging tissue types for this application. We describe steps to generate skin wounds in mice, followed by tissue harvest and oligonucleotide barcoding. This protocol is also applicable to other species including rats, pigs, and humans. For complete details on the use and execution of this protocol, please refer to Stoeckius et al. (2018), Galiano et al. (2004), and Mascharak et al. (2022)..
• Jiang, Y., Trotsyuk, A. A., Niu, S., Henn, D., Chen, K., Shih, C. C., Larson, M. R., Mermin-Bunnell, A. M., Mittal, S., Lai, J. C., Saberi, A., Beard, E., Jing, S., Zhong, D., Steele, S. R., Sun, K., Jain, T., Zhao, E., Neimeth, C. R., , Viana, W. G., et al. (2022). Wireless, closed-loop, smart bandage with integrated sensors and stimulators for advanced wound care and accelerated healing. Nature biotechnology.
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  '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.
• Mascharak, S., Talbott, H. E., Januszyk, M., Griffin, M., Chen, K., Davitt, M. F., Demeter, J., Henn, D., Bonham, C. A., Foster, D. S., Mooney, N., Cheng, R., Jackson, P. K., Wan, D. C., Gurtner, G. C., & Longaker, M. T. (2022). Multi-omic analysis reveals divergent molecular events in scarring and regenerative wound healing. Cell stem cell, 29(2), 315-327.e6.
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  Regeneration is the holy grail of tissue repair, but skin injury typically yields fibrotic, non-functional scars. Developing pro-regenerative therapies requires rigorous understanding of the molecular progression from injury to fibrosis or regeneration. Here, we report the divergent molecular events driving skin wound cells toward scarring or regenerative fates. We profile scarring versus YAP-inhibition-induced wound regeneration at the transcriptional (single-cell RNA sequencing), protein (timsTOF proteomics), and tissue (extracellular matrix ultrastructural analysis) levels. Using cell-surface barcoding, we integrate these data to reveal fibrotic and regenerative "molecular trajectories" of healing. We show that disrupting YAP mechanotransduction yields regenerative repair by fibroblasts with activated Trps1 and Wnt signaling. Finally, via in vivo gene knockdown and overexpression in wounds, we identify Trps1 as a key regulatory gene that is necessary and partially sufficient for wound regeneration. Our findings serve as a multi-omic map of wound regeneration and could have therapeutic implications for pathologic fibroses.
• Padmanabhan, J., Liu, F. C., Sivaraj, D., Henn, D., Chen, K., Simon, D. R., Barrera, J. A., & Gurtner, G. C. (2022). Two Independent Capsules Surrounding a Single Textured Implant in Ehlers-Danlos Syndrome. Plastic and reconstructive surgery. Global open, 10(8), e4470.
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  Textured breast implants are associated with prolonged inflammation leading to increased risk for complications such as the development of anaplastic large cell lymphoma. The underlying molecular mechanisms that drive increased inflammation toward textured implants (compared with smooth implants) remain poorly understood. Here, we present the first known case of a patient with Ehlers-Danlos syndrome (EDS) who developed two independent fibrotic capsules around a single textured silicone implant. The patient was found to have one internal capsule tightly adherent to the implant and a second external capsule that was attached to the surrounding tissue. We observed that the internal implant-adherent capsule was composed of a highly aligned and dense collagen network, completely atypical for EDS and indicative of a high mechanical stress environment. In contrast, the external nonadherent capsule, which primarily interacted with the smooth surface of the internal capsule, displayed disorganized collagen fibers with no discernible alignment, classic for EDS. Remarkably, we found that the internal capsule displayed high activation of monocyte chemoattractant protein-1, a mechanoresponsive inflammatory mediator that was not elevated in the disorganized external capsule. Taken together, these findings demonstrate that the tight adhesion between the textured implant surface and the internal capsule creates a high mechanical stress environment, which is responsible for the increased local inflammation observed in the internal capsule. This unique case demonstrates that mechanical stress is able to override genetic defects locally in collagen organization and directly connects the textured surface of implants to prolonged inflammation.
• Sivaraj, D., Fischer, K. S., Kim, T. S., Chen, K., Tigchelaar, S. S., Trotsyuk, A. A., Gurtner, G. C., Lee, G. K., Henn, D., & Nazerali, R. S. (2022). Outcomes of Biosynthetic and Synthetic Mesh in Ventral Hernia Repair. Plastic and reconstructive surgery. Global open, 10(12), e4707.
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  The introduction of mesh for reinforcement of ventral hernia repair (VHR) led to a significant reduction in hernia recurrence rates. However, it remains controversial whether synthetic or biologic mesh leads to superior outcomes. Recently, hybrid mesh consisting of reinforced biosynthetic ovine rumen (RBOR) has been developed and aims to combine the advantages of biologic and synthetic mesh; however, outcomes after VHR with RBOR have not yet been compared with the standard of care.
• Sivaraj, D., Henn, D., Fischer, K. S., Kim, T. S., Black, C. K., Lin, J. Q., Barrera, J. A., Leeolou, M. C., Makarewicz, N. S., Chen, K., Perrault, D. P., Gurtner, G. C., Lee, G. K., & Nazerali, R. (2022). Reinforced Biologic Mesh Reduces Postoperative Complications Compared to Biologic Mesh after Ventral Hernia Repair. Plastic and reconstructive surgery. Global open, 10(2), e4083.
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  The use of biologic mesh to reinforce the abdominal wall in ventral hernia repair has been proposed as a viable alternative to synthetic mesh, particularly for high-risk patients and in contaminated settings. However, a comparison of clinical outcomes between the currently available biologic mesh types has yet to be performed.
• Sivaraj, D., Padmanabhan, J., Chen, K., Henn, D., Noishiki, C., Trotsyuk, A. A., Kussie, H. C., Leeolou, M. C., Magbual, N. J., Andrikopoulos, S., Perrault, D. P., Barrera, J. A., Januszyk, M., & Gurtner, G. C. (2022). IQGAP1-mediated mechanical signaling promotes the foreign body response to biomedical implants. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 36(2), e22007.
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  The aim of this study was to further elucidate the molecular mechanisms that mediate pathologic foreign body response (FBR) to biomedical implants. The longevity of biomedical implants is limited by the FBR, which leads to implant failure and patient morbidity. Since the specific molecular mechanisms underlying fibrotic responses to biomedical implants have yet to be fully described, there are currently no targeted approaches to reduce pathologic FBR. We utilized proteomics analysis of human FBR samples to identify potential molecular targets for therapeutic inhibition of FBR. We then employed a murine model of FBR to further evaluate the role of this potential target. We performed histological and immunohistochemical analysis on the murine FBR capsule tissue, as well as single-cell RNA sequencing (scRNA-seq) on cells isolated from the capsules. We identified IQ motif containing GTPase activating protein 1 (IQGAP1) as the most promising of several targets, serving as a central molecular mediator in human and murine FBR compared to control subcutaneous tissue. IQGAP1-deficient mice displayed a significantly reduced FBR compared to wild-type mice as evidenced by lower levels of collagen deposition and maturity. Our scRNA-seq analysis revealed that decreasing IQGAP1 resulted in diminished transcription of mechanotransduction, inflammation, and fibrosis-related genes, which was confirmed on the protein level with immunofluorescent staining. The deficiency of IQGAP1 significantly attenuates FBR by deactivating downstream mechanotransduction signaling, inflammation, and fibrotic pathways. IQGAP1 may be a promising target for rational therapeutic design to mitigate pathologic FBR around biomedical implants.
• Titan, A. L., Davitt, M., Foster, D., Salhotra, A., Menon, S., Chen, K., Fahy, E., Lopez, M., Jones, R. E., Baiu, I., Burcham, A., Januszyk, M., Gurtner, G., Fox, P., Chan, C., Quarto, N., & Longaker, M. (2022). Partial Tendon Injury at the Tendon-to-Bone Enthesis Activates Skeletal Stem Cells. Stem cells translational medicine, 11(7), 715-726.
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  The tendon enthesis plays a critical role in facilitating movement and reducing stress within joints. Partial enthesis injuries heal in a mechanically inferior manner and never achieve healthy tissue function. The cells responsible for tendon-to-bone healing remain incompletely characterized and their origin is unknown. Here, we evaluated the putative role of mouse skeletal stem cells (mSSCs) in the enthesis after partial-injury. We found that mSSCs were present at elevated levels within the enthesis following injury and that these cells downregulated TGFβ signaling pathway elements at both the RNA and protein levels. Exogenous application of TGFβ post-injury led to a reduced mSSC response and impaired healing, whereas treatment with a TGFβ inhibitor (SB43154) resulted in a more robust mSSC response. Collectively, these data suggest that mSSCs may augment tendon-to-bone healing by dampening the effects of TGFβ signaling within the mSSC niche.
• Trotsyuk, A. A., Chen, K., Hyung, S., Ma, K. C., Henn, D., Mermin-Bunnell, A. M., Mittal, S., Padmanabhan, J., Larson, M. R., Steele, S. R., Sivaraj, D., Bonham, C. A., Noishiki, C., Rodrigues, M., Jiang, Y., Jing, S., Niu, S., Chattopadhyay, A., Perrault, D. P., , Leeolou, M. C., et al. (2022). Inhibiting Fibroblast Mechanotransduction Modulates Severity of Idiopathic Pulmonary Fibrosis. Advances in wound care, 11(10), 511-523.
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  Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease that affects 63 in every 100,000 Americans. Its etiology remains unknown, although inflammatory pathways appear to be important. Given the dynamic environment of the lung, we examined the significance of mechanotransduction on both inflammatory and fibrotic signaling during IPF. Mechanotransduction pathways have not been thoroughly examined in the context of lung disease, and pharmacologic approaches for IPF do not currently target these pathways. The interplay between mechanical strain and inflammation in pulmonary fibrosis remains incompletely understood. In this study, we used conditional KO mice to block mechanotransduction by knocking out Focal Adhesion Kinase (FAK) expression in fibroblasts, followed by induction of pulmonary fibrosis using bleomycin. We examined both normal human and human IPF fibroblasts and used immunohistochemistry, quantitative real-time polymerase chain reaction, and Western Blot to evaluate the effects of FAK inhibitor (FAK-I) on modulating fibrotic and inflammatory genes. Our data indicate that the deletion of FAK in mice reduces expression of fibrotic and inflammatory genes in lungs. Similarly, mechanical straining in normal human lung fibroblasts activates inflammatory and fibrotic pathways. The FAK inhibition decreases these signals but has a less effect on IPF fibroblasts as compared with normal human fibroblasts. Administering FAK-I at early stages of fibrosis may attenuate the FAK-mediated fibrotic response pathway in IPF, potentially mediating disease progression.
• Barrera, J. A., Trotsyuk, A. A., Maan, Z. N., Bonham, C. A., Larson, M. R., Mittermiller, P. A., Henn, D., Chen, K., Mays, C. J., Mittal, S., Mermin-Bunnell, A. M., Sivaraj, D., Jing, S., Rodrigues, M., Kwon, S. H., Noishiki, C., Padmanabhan, J., Jiang, Y., Niu, S., , Inayathullah, M., et al. (2021). Adipose-Derived Stromal Cells Seeded in Pullulan-Collagen Hydrogels Improve Healing in Murine Burns. Tissue engineering. Part A, 27(11-12), 844-856.
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  Burn scars and scar contractures cause significant morbidity for patients. Recently, cell-based therapies have been proposed as an option for improving healing and reducing scarring after burn injury, through their known proangiogenic and immunomodulatory paracrine effects. Our laboratory has developed a pullulan-collagen hydrogel that, when seeded with mesenchymal stem cells (MSCs), improves cell viability and augments their proangiogenic capacity . Concurrently, recent research suggests that prospective isolation of cell subpopulations with desirable transcriptional profiles can be used to further improve cell-based therapies. In this study, we examined whether adipose-derived stem cell (ASC)-seeded hydrogels could improve wound healing following thermal injury using a murine contact burn model. Partial thickness contact burns were created on the dorsum of mice. On days 5 and 10 following injury, burns were debrided and received either ASC hydrogel, ASC injection alone, hydrogel alone, or no treatment. On days 10 and 25, burns were harvested for histologic and molecular analysis. This experiment was repeated using CD26/CD55 FACS-enriched ASCs to further evaluate the regenerative potential of ASCs in wound healing. ASC hydrogel-treated burns demonstrated accelerated time to reepithelialization, greater vascularity, and increased expression of the proangiogenic genes MCP-1, VEGF, and SDF-1 at both the mRNA and protein level. Expression of the profibrotic gene and proinflammatory gene was downregulated in ASC hydrogel-treated burns. ASC hydrogel-treated burns exhibited reduced scar area compared to hydrogel-treated and control wounds, with equivalent scar density. CD26/CD55 ASC hydrogel treatment resulted in accelerated healing, increased dermal appendage count, and improved scar quality with a more reticular collagen pattern. Here we find that ASC hydrogel therapy is effective for treating burns, with demonstrated proangiogenic, fibromodulatory, and immunomodulatory effects. Enrichment for CD26/CD55 ASCs has additive benefits for tissue architecture and collagen remodeling postburn injury. Research is ongoing to further facilitate clinical translation of this promising therapeutic approach. Impact statement Burns remain a significant public health burden. Stem cell therapy has gained attention as a promising approach for treating burns. We have developed a pullulan-collagen biomimetic hydrogel scaffold that can be seeded with adipose-derived stem cells (ASCs). We assessed the delivery and activity of our scaffold in a murine contact burn model. Our results suggest that localized delivery of ASC hydrogel treatment is a promising approach for the treatment of burn wounds, with the potential for rapid clinical translation. We believe our work will have broad implications for both hydrogel therapeutics and regenerative medicine and will be of interest to the general scientific community.
• Chen, K., Kwon, S. H., Henn, D., Kuehlmann, B. A., Tevlin, R., Bonham, C. A., Griffin, M., Trotsyuk, A. A., Borrelli, M. R., Noishiki, C., Padmanabhan, J., Barrera, J. A., Maan, Z. N., Dohi, T., Mays, C. J., Greco, A. H., Sivaraj, D., Lin, J. Q., Fehlmann, T., , Mermin-Bunnell, A. M., et al. (2021). Disrupting biological sensors of force promotes tissue regeneration in large organisms. Nature communications, 12(1), 5256.
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  Tissue repair and healing remain among the most complicated processes that occur during postnatal life. Humans and other large organisms heal by forming fibrotic scar tissue with diminished function, while smaller organisms respond with scarless tissue regeneration and functional restoration. Well-established scaling principles reveal that organism size exponentially correlates with peak tissue forces during movement, and evolutionary responses have compensated by strengthening organ-level mechanical properties. How these adaptations may affect tissue injury has not been previously examined in large animals and humans. Here, we show that blocking mechanotransduction signaling through the focal adhesion kinase pathway in large animals significantly accelerates wound healing and enhances regeneration of skin with secondary structures such as hair follicles. In human cells, we demonstrate that mechanical forces shift fibroblasts toward pro-fibrotic phenotypes driven by ERK-YAP activation, leading to myofibroblast differentiation and excessive collagen production. Disruption of mechanical signaling specifically abrogates these responses and instead promotes regenerative fibroblast clusters characterized by AKT-EGR1.
• Foster, D. S., Januszyk, M., Yost, K. E., Chinta, M. S., Gulati, G. S., Nguyen, A. T., Burcham, A. R., Salhotra, A., Ransom, R. C., Henn, D., Chen, K., Mascharak, S., Tolentino, K., Titan, A. L., Jones, R. E., da Silva, O., Leavitt, W. T., Marshall, C. D., des Jardins-Park, H. E., , Hu, M. S., et al. (2021). Integrated spatial multiomics reveals fibroblast fate during tissue repair. Proceedings of the National Academy of Sciences of the United States of America, 118(41).
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  In the skin, tissue injury results in fibrosis in the form of scars composed of dense extracellular matrix deposited by fibroblasts. The therapeutic goal of regenerative wound healing has remained elusive, in part because principles of fibroblast programming and adaptive response to injury remain incompletely understood. Here, we present a multimodal -omics platform for the comprehensive study of cell populations in complex tissue, which has allowed us to characterize the cells involved in wound healing across both time and space. We employ a stented wound model that recapitulates human tissue repair kinetics and multiple Rainbow transgenic lines to precisely track fibroblast fate during the physiologic response to skin injury. Through integrated analysis of single cell chromatin landscapes and gene expression states, coupled with spatial transcriptomic profiling, we are able to impute fibroblast epigenomes with temporospatial resolution. This has allowed us to reveal potential mechanisms controlling fibroblast fate during migration, proliferation, and differentiation following skin injury, and thereby reexamine the canonical phases of wound healing. These findings have broad implications for the study of tissue repair in complex organ systems.
• Griffin, M. F., Borrelli, M. R., Garcia, J. T., Januszyk, M., King, M., Lerbs, T., Cui, L., Moore, A. L., Shen, A. H., Mascharak, S., Diaz Deleon, N. M., Adem, S., Taylor, W. L., desJardins-Park, H. E., Gastou, M., Patel, R. A., Duoto, B. A., Sokol, J., Wei, Y., , Foster, D., et al. (2021). JUN promotes hypertrophic skin scarring via CD36 in preclinical in vitro and in vivo models. Science translational medicine, 13(609), eabb3312.
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  Pathologic skin scarring presents a vast economic and medical burden. Unfortunately, the molecular mechanisms underlying scar formation remain to be elucidated. We used a hypertrophic scarring (HTS) mouse model in which is overexpressed globally or specifically in α-smooth muscle or collagen type I–expressing cells to cause excessive extracellular matrix deposition by skin fibroblasts in the skin after wounding. overexpression triggered dermal fibrosis by modulating distinct fibroblast subpopulations within the wound, enhancing reticular fibroblast numbers, and decreasing lipofibroblasts. Analysis of human scars further revealed that JUN is highly expressed across the wide spectrum of scars, including HTS and keloids. CRISPR-Cas9–mediated JUN deletion in human HTS fibroblasts combined with epigenomic and transcriptomic analysis of both human and mouse HTS fibroblasts revealed that JUN initiates fibrosis by regulating . Blocking CD36 with salvianolic acid B or CD36 knockout model counteracted JUN-mediated fibrosis efficacy in both human fibroblasts and mouse wounds. In summary, JUN is a critical regulator of pathological skin scarring, and targeting its downstream effector CD36 may represent a therapeutic strategy against scarring.
• Henn, D., Chen, K., Fehlmann, T., Trotsyuk, A. A., Sivaraj, D., Maan, Z. N., Bonham, C. A., Barrera, J. A., Mays, C. J., Greco, A. H., Moortgat Illouz, S. E., Lin, J. Q., Steele, S. R., Foster, D. S., Padmanabhan, J., Momeni, A., Nguyen, D., Wan, D. C., Kneser, U., , Januszyk, M., et al. (2021). Xenogeneic skin transplantation promotes angiogenesis and tissue regeneration through activated Trem2 macrophages. Science advances, 7(49), eabi4528.
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  Skin allo- and xenotransplantation are the standard treatment for major burns when donor sites for autografts are not available. The relationship between the immune response to foreign grafts and their impact on wound healing has not been fully elucidated. Here, we investigated changes in collagen architecture after xenogeneic implantation of human biologic scaffolds. We show that collagen deposition in response to the implantation of human split-thickness skin grafts (hSTSGs) containing live cells recapitulates normal skin architecture, whereas human acellular dermal matrix (ADM) grafts led to a fibrotic collagen deposition. We show that macrophage differentiation in response to hSTSG implantation is driven toward regenerative Trem2 subpopulations and found that hydrogel delivery of these cells significantly accelerated wound closure. Our study identifies the preclinical therapeutic potential of Trem2 macrophages to mitigate fibrosis and promote wound healing, providing a novel effective strategy to develop advanced cell therapies for complex wounds.
• Henn, D., Sivaraj, D., Barrera, J. A., Lin, J. Q., Chattopadhyay, A., Maan, Z. N., Chen, K., Nguyen, A., Cheesborough, J., Gurtner, G. C., Lee, G. K., & Nazerali, R. (2021). The Plane of Mesh Placement Does Not Impact Abdominal Donor Site Complications in Microsurgical Breast Reconstruction. Annals of plastic surgery, 87(5), 542-546.
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  Reinforcement of the abdominal wall with synthetic mesh in autologous breast reconstruction using abdominal free tissue transfer decreases the risk of bulging and herniation. However, the impact of the plane of mesh placement on donor site complications has not yet been investigated.
• Lavin, C. V., Abbas, D. B., Fahy, E. J., Lee, D. K., Griffin, M., Diaz Deleon, N. M., Mascharak, S., Chen, K., Momeni, A., Gurtner, G. C., Longaker, M. T., & Wan, D. C. (2021). A comparative analysis of deferoxamine treatment modalities for dermal radiation-induced fibrosis. Journal of cellular and molecular medicine, 25(21), 10028-10038.
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  The iron chelator, deferoxamine (DFO), has been shown to potentially improve dermal radiation-induced fibrosis (RIF) in mice through increased angiogenesis and reduced oxidative damage. This preclinical study evaluated the efficacy of two DFO administration modalities, transdermal delivery and direct injection, as well as temporal treatment strategies in relation to radiation therapy to address collateral soft tissue fibrosis. The dorsum of CD-1 nude mice received 30 Gy radiation, and DFO (3 mg) was administered daily via patch or injection. Treatment regimens were prophylactic, during acute recovery, post-recovery, or continuously throughout the experiment (n = 5 per condition). Measures included ROS-detection, histology, biomechanics and vascularity changes. Compared with irradiated control skin, DFO treatment decreased oxidative damage, dermal thickness and collagen content, and increased skin elasticity and vascularity. Metrics of improvement in irradiated skin were most pronounced with continuous transdermal delivery of DFO. In summary, DFO administration reduces dermal fibrosis induced by radiation. Although both treatment modalities were efficacious, the transdermal delivery showed greater effect than injection for each temporal treatment strategy. Interestingly, the continuous patch group was more similar to normal skin than to irradiated control skin by most measures, highlighting a promising approach to address detrimental collateral soft tissue injury following radiation therapy.
• Leeolou, M. C., Perrault, D. P., Sivaraj, D., Chang, A. L., Chen, K., Trotsyuk, A. A., Padmanabhan, J., & Gurtner, G. C. (2021). A rare case of infection in California. JAAD case reports, 17, 55-57.
• Maan, Z. N., Rinkevich, Y., Barrera, J., Chen, K., Henn, D., Foster, D., Bonham, C. A., Padmanabhan, J., Sivaraj, D., Duscher, D., Hu, M., Yan, K., Januszyk, M., Longaker, M. T., Weissman, I. L., & Gurtner, G. C. (2021). Epidermal-Derived Hedgehog Signaling Drives Mesenchymal Proliferation during Digit Tip Regeneration. Journal of clinical medicine, 10(18).
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  Hand injuries often result in significant functional impairments and are rarely completely restored. The spontaneous regeneration of injured appendages, which occurs in salamanders and newts, for example, has been reported in human fingertips after distal amputation, but this type of regeneration is rare in mammals and is incompletely understood. Here, we study fingertip regeneration by amputating murine digit tips, either distally to initiate regeneration, or proximally, causing fibrosis. Using an unbiased microarray analysis, we found that digit tip regeneration is significantly associated with hair follicle differentiation, Wnt, and sonic hedgehog (SHH) signaling pathways. Viral over-expression and genetic knockouts showed the functional significance of these pathways during regeneration. Using transgenic reporter mice, we demonstrated that, while both canonical Wnt and HH signaling were limited to epidermal tissues, downstream hedgehog signaling (through Gli) occurred in mesenchymal tissues. These findings reveal a mechanism for epidermal/mesenchyme interactions, governed by canonical hedgehog signaling, during digit regeneration. Further research into these pathways could lead to improved therapeutic outcomes after hand injuries in humans.
• Mascharak, S., desJardins-Park, H. E., Davitt, M. F., Griffin, M., Borrelli, M. R., Moore, A. L., Chen, K., Duoto, B., Chinta, M., Foster, D. S., Shen, A. H., Januszyk, M., Kwon, S. H., Wernig, G., Wan, D. C., Lorenz, H. P., Gurtner, G. C., & Longaker, M. T. (2021). Preventing activation in fibroblasts yields wound regeneration without scarring. Science (New York, N.Y.), 372(6540).
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  Skin scarring, the end result of adult wound healing, is detrimental to tissue form and function. lineage-positive fibroblasts (EPFs) are known to function in scarring, but lineage-negative fibroblasts (ENFs) remain poorly characterized. Using cell transplantation and transgenic mouse models, we identified a dermal ENF subpopulation that gives rise to postnatally derived EPFs by activating expression during adult wound healing. By studying ENF responses to substrate mechanics, we found that mechanical tension drives activation via canonical mechanotransduction signaling. Finally, we showed that blocking mechanotransduction signaling with either verteporfin, an inhibitor of Yes-associated protein (YAP), or fibroblast-specific transgenic YAP knockout prevents activation and promotes wound regeneration by ENFs, with recovery of skin appendages, ultrastructure, and mechanical strength. This finding suggests that there are two possible outcomes to postnatal wound healing: a fibrotic response (EPF-mediated) and a regenerative response (ENF-mediated).
• Sivaraj, D., Chen, K., Chattopadhyay, A., Henn, D., Wu, W., Noishiki, C., Magbual, N. J., Mittal, S., Mermin-Bunnell, A. M., Bonham, C. A., Trotsyuk, A. A., Barrera, J. A., Padmanabhan, J., Januszyk, M., & Gurtner, G. C. (2021). Hydrogel Scaffolds to Deliver Cell Therapies for Wound Healing. Frontiers in bioengineering and biotechnology, 9, 660145.
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  Cutaneous wounds are a growing global health burden as a result of an aging population coupled with increasing incidence of diabetes, obesity, and cancer. Cell-based approaches have been used to treat wounds due to their secretory, immunomodulatory, and regenerative effects, and recent studies have highlighted that delivery of stem cells may provide the most benefits. Delivering these cells to wounds with direct injection has been associated with low viability, transient retention, and overall poor efficacy. The use of bioactive scaffolds provides a promising method to improve cell therapy delivery. Specifically, hydrogels provide a physiologic microenvironment for transplanted cells, including mechanical support and protection from native immune cells, and cell-hydrogel interactions may be tailored based on specific tissue properties. In this review, we describe the current and future directions of various cell therapies and usage of hydrogels to deliver these cells for wound healing applications.
• Titan, A. L., Fahy, E., Chen, K., Foster, D. S., Bennett-Kennett, R., Dauskardt, R. H., Gurtner, G. C., Chang, J., Fox, P. M., & Longaker, M. T. (2021). Proceed with Caution: Mouse Deep Digit Flexor Tendon Injury Model. Plastic and reconstructive surgery. Global open, 9(1), e3359.
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  The purpose of this study was to determine the feasibility of using mouse models for translational study of flexor tendon repair and reconstruction.
• Henn, D., Abu-Halima, M., Kahraman, M., Falkner, F., Fischer, K. S., Barrera, J. A., Chen, K., Gurtner, G. C., Keller, A., Kneser, U., Meese, E., & Schmidt, V. J. (2020). A multivariable miRNA signature delineates the systemic hemodynamic impact of arteriovenous shunt placement in a pilot study. Scientific reports, 10(1), 21809.
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  Arteriovenous (AV) fistulas for hemodialysis can lead to cardiac volume loading and increased serum brain natriuretic peptide (BNP) levels. Whether short-term AV loop placement in patients undergoing microsurgery has an impact on cardiac biomarkers and circulating microRNAs (miRNAs), potentially indicating an increased hemodynamic risk, remains elusive. Fifteen patients underwent AV loop placement with delayed free flap anastomosis for microsurgical reconstructions of lower extremity soft-tissue defects. N-terminal pro-BNP (NT-proBNP), copeptin (CT-proAVP), and miRNA expression profiles were determined in the peripheral blood before and after AV loop placement. MiRNA expression in the blood was correlated with miRNA expression from AV loop vascular tissue. Serum NT-proBNP and copeptin levels exceeded the upper reference limit after AV loop placement, with an especially strong NT-proBNP increase in patients with preexistent cardiac diseases. A miRNA signature of 4 up-regulated (miR-3198, miR-3127-5p, miR-1305, miR-1288-3p) and 2 down-regulated miRNAs (miR30a-5p, miR-145-5p) which are related to cardiovascular physiology, showed a significant systemic deregulation in blood and venous tissue after AV loop placement. AV loop placement causes serum elevations of NT-proBNP, copeptin as well as specific circulating miRNAs, indicating a potentially increased hemodynamic risk for patients with cardiovascular comorbidities, if free flap anastomosis is delayed.
• Henn, D., Chen, K., Fischer, K., Rauh, A., Barrera, J. A., Kim, Y. J., Martin, R. A., Hannig, M., Niedoba, P., Reddy, S. K., Mao, H. Q., Kneser, U., Gurtner, G. C., Sacks, J. M., & Schmidt, V. J. (2020). Tissue Engineering of Axially Vascularized Soft-Tissue Flaps with a Poly-(ɛ-Caprolactone) Nanofiber-Hydrogel Composite. Advances in wound care, 9(7), 365-377.
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  To develop a novel approach for tissue engineering of soft-tissue flaps suitable for free microsurgical transfer, using an injectable nanofiber hydrogel composite (NHC) vascularized by an arteriovenous (AV) loop. A rat AV loop model was used for tissue engineering of vascularized soft-tissue flaps. NHC or collagen-elastin (CE) scaffolds were implanted into isolation chambers together with an AV loop and explanted after 15 days. Saphenous veins were implanted into the scaffolds as controls. Neoangiogenesis, ultrastructure, and protein expression of SYNJ2BP, EPHA2, and FOXC1 were analyzed by immunohistochemistry and compared between the groups. Rheological properties were compared between the two scaffolds and native human adipose tissue. A functional neovascularization was evident in NHC flaps with its amount being comparable with CE flaps. Scanning electron microscopy revealed a strong mononuclear cell infiltration along the nanofibers in NHC flaps and a trend toward higher fiber alignment compared with CE flaps. SYNJ2BP and EPHA2 expression in endothelial cells (ECs) was lower in NHC flaps compared with CE flaps, whereas FOXC1 expression was increased in NHC flaps. Compared with the stiffer CE flaps, the NHC flaps showed similar rheological properties to native human adipose tissue. This is the first study to demonstrate the feasibility of tissue engineering of soft-tissue flaps with similar rheological properties as human fat, suitable for microsurgical transfer using an injectable nanofiber hydrogel composite. The injectable NHC scaffold is suitable for tissue engineering of axially vascularized soft-tissue flaps with a solid neovascularization, strong cellular infiltration, and biomechanical properties similar to human fat. Our data indicate that SYNJ2BP, EPHA2, and FOXC1 are involved in AV loop-associated angiogenesis and that the scaffold material has an impact on protein expression in ECs.
• Henn, D., Chen, K., Maan, Z. N., Greco, A. H., Moortgat Illouz, S. E., Bonham, C. A., Barrera, J. A., Trotsyuk, A. A., Padmanabhan, J., Momeni, A., Wan, D. C., Nguyen, D., Januszyk, M., & Gurtner, G. C. (2020). Cryopreserved human skin allografts promote angiogenesis and dermal regeneration in a murine model. International wound journal, 17(4), 925-936.
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  Cryopreserved human skin allografts (CHSAs) are used for the coverage of major burns when donor sites for autografts are insufficiently available and have clinically shown beneficial effects on chronic non-healing wounds. However, the biologic mechanisms behind the regenerative properties of CHSA remain elusive. Furthermore, the impact of cryopreservation on the immunogenicity of CHSA has not been thoroughly investigated and raised concerns with regard to their clinical application. To investigate the importance and fate of living cells, we compared cryopreserved CHSA with human acellular dermal matrix (ADM) grafts in which living cells had been removed by chemical processing. Both grafts were subcutaneously implanted into C57BL/6 mice and explanted after 1, 3, 7, and 28 days (n = 5 per group). A sham surgery where no graft was implanted served as a control. Transmission electron microscopy (TEM) and flow cytometry were used to characterise the ultrastructure and cells within CHSA before implantation. Immunofluorescent staining of tissue sections was used to determine the immune reaction against the implanted grafts, the rate of apoptotic cells, and vascularisation as well as collagen content of the overlaying murine dermis. Digital quantification of collagen fibre alignment on tissue sections was used to quantify the degree of fibrosis within the murine dermis. A substantial population of live human cells with intact organelles was identified in CHSA prior to implantation. Subcutaneous pockets with implanted xenografts or ADMs healed without clinically apparent rejection and with a similar cellular immune response. CHSA implantation largely preserved the cellularity of the overlying murine dermis, whereas ADM was associated with a significantly higher rate of cellular apoptosis, identified by cleaved caspase-3 staining, and a stronger dendritic cell infiltration of the murine dermis. CHSA was found to induce a local angiogenic response, leading to significantly more vascularisation of the murine dermis compared with ADM and sham surgery on day 7. By day 28, aggregate collagen-1 content within the murine dermis was greater following CHSA implantation compared with ADM. Collagen fibre alignment of the murine dermis, correlating with the degree of fibrosis, was significantly greater in the ADM group, whereas CHSA maintained the characteristic basket weave pattern of the native murine dermis. Our data indicate that CHSAs promote angiogenesis and collagen-1 production without eliciting a significant fibrotic response in a xenograft model. These findings may provide insight into the beneficial effects clinically observed after treatment of chronic wounds and burns with CHSA.
• Januszyk, M., Chen, K., Henn, D., Foster, D. S., Borrelli, M. R., Bonham, C. A., Sivaraj, D., Wagh, D., Longaker, M. T., Wan, D. C., & Gurtner, G. C. (2020). Characterization of Diabetic and Non-Diabetic Foot Ulcers Using Single-Cell RNA-Sequencing. Micromachines, 11(9).
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  Recent advances in high-throughput single-cell sequencing technologies have led to their increasingly widespread adoption for clinical applications. However, challenges associated with tissue viability, cell yield, and delayed time-to-capture have created unique obstacles for data processing. Chronic wounds, in particular, represent some of the most difficult target specimens, due to the significant amount of fibrinous debris, extracellular matrix components, and non-viable cells inherent in tissue routinely obtained from debridement. Here, we examined the feasibility of single cell RNA sequencing (scRNA-seq) analysis to evaluate human chronic wound samples acquired in the clinic, subjected to prolonged cold ischemia time, and processed without FACS sorting. Wound tissue from human diabetic and non-diabetic plantar foot ulcers were evaluated using an optimized 10X Genomics scRNA-seq platform and analyzed using a modified data pipeline designed for low-yield specimens. Cell subtypes were identified informatically and their distributions and transcriptional programs were compared between diabetic and non-diabetic tissue. 139,000 diabetic and non-diabetic wound cells were delivered for 10X capture after either 90 or 180 min of cold ischemia time. cDNA library concentrations were 858.7 and 364.7 pg/µL, respectively, prior to sequencing. Among all barcoded fragments, we found that 83.5% successfully aligned to the human transcriptome and 68% met the minimum cell viability threshold. The average mitochondrial mRNA fraction was 8.5% for diabetic cells and 6.6% for non-diabetic cells, correlating with differences in cold ischemia time. A total of 384 individual cells were of sufficient quality for subsequent analyses; from this cell pool, we identified transcriptionally-distinct cell clusters whose gene expression profiles corresponded to fibroblasts, keratinocytes, neutrophils, monocytes, and endothelial cells. Fibroblast subpopulations with differing fibrotic potentials were identified, and their distributions were found to be altered in diabetic vs. non-diabetic cells. scRNA-seq of clinical wound samples can be achieved using minor modifications to standard processing protocols and data analysis methods. This simple approach can capture widespread transcriptional differences between diabetic and non-diabetic tissue obtained from matched wound locations.
• Kosaric, N., Srifa, W., Bonham, C. A., Kiwanuka, H., Chen, K., Kuehlmann, B. A., Maan, Z. N., Noishiki, C., Porteus, M. H., Longaker, M. T., & Gurtner, G. C. (2020). Macrophage Subpopulation Dynamics Shift following Intravenous Infusion of Mesenchymal Stromal Cells. Molecular therapy : the journal of the American Society of Gene Therapy, 28(9), 2007-2022.
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  Intravenous infusion of mesenchymal stromal cells (MSCs) is thought to be a viable treatment for numerous disorders. Although the intrinsic immunosuppressive ability of MSCs has been credited for this therapeutic effect, their exact impact on endogenous tissue-resident cells following delivery has not been clearly characterized. Moreover, multiple studies have reported pulmonary sequestration of MSCs upon intravenous delivery. Despite substantial efforts to improve MSC homing, it remains unclear whether MSC migration to the site of injury is necessary to achieve a therapeutic effect. Using a murine excisional wound healing model, we offer an explanation of how sequestered MSCs improve healing through their systemic impact on macrophage subpopulations. We demonstrate that infusion of MSCs leads to pulmonary entrapment followed by rapid clearance, but also significantly accelerates wound closure. Using single-cell RNA sequencing of the wound, we show that following MSC delivery, innate immune cells, particularly macrophages, exhibit distinctive transcriptional changes. We identify the appearance of a pro-angiogenic CD9 macrophage subpopulation, whose induction is mediated by several proteins secreted by MSCs, including COL6A1, PRG4, and TGFB3. Our findings suggest that MSCs do not need to act locally to induce broad changes in the immune system and ultimately treat disease.
• Kwon, S. H., Barrera, J. A., Noishiki, C., Chen, K., Henn, D., Sheckter, C. C., & Gurtner, G. C. (2020). Current and Emerging Topical Scar Mitigation Therapies for Craniofacial Burn Wound Healing. Frontiers in physiology, 11, 916.
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  Burn injury in the craniofacial region causes significant health and psychosocial consequences and presents unique reconstructive challenges. Healing of severely burned skin and underlying soft tissue is a dynamic process involving many pathophysiological factors, often leading to devastating outcomes such as the formation of hypertrophic scars and debilitating contractures. There are limited treatment options currently used for post-burn scar mitigation but recent advances in our knowledge of the cellular and molecular wound and scar pathophysiology have allowed for development of new treatment concepts. Clinical effectiveness of these experimental therapies is currently being evaluated. In this review, we discuss current topical therapies for craniofacial burn injuries and emerging new therapeutic concepts that are highly translational.
• Chen, K., Hu, X., Blemker, S. S., & Holmes, J. W. (2018). Multiscale computational model of Achilles tendon wound healing: Untangling the effects of repair and loading. PLoS computational biology, 14(12), e1006652.
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  Mechanical stimulation of the healing tendon is thought to regulate scar anisotropy and strength and is relatively easy to modulate through physical therapy. However, in vivo studies of various loading protocols in animal models have produced mixed results. To integrate and better understand the available data, we developed a multiscale model of rat Achilles tendon healing that incorporates the effect of changes in the mechanical environment on fibroblast behavior, collagen deposition, and scar formation. We modified an OpenSim model of the rat right hindlimb to estimate physiologic strains in the lateral/medial gastrocnemius and soleus musculo-tendon units during loading and unloading conditions. We used the tendon strains as inputs to a thermodynamic model of stress fiber dynamics that predicts fibroblast alignment, and to determine local collagen synthesis rates according to a response curve derived from in vitro studies. We then used an agent-based model (ABM) of scar formation to integrate these cell-level responses and predict tissue-level collagen alignment and content. We compared our model predictions to experimental data from ten different studies. We found that a single set of cellular response curves can explain features of observed tendon healing across a wide array of reported experiments in rats-including the paradoxical finding that repairing transected tendon reverses the effect of loading on alignment-without fitting model parameters to any data from those experiments. The key to these successful predictions was simulating the specific loading and surgical protocols to predict tissue-level strains, which then guided cellular behaviors according to response curves based on in vitro experiments. Our model results provide a potential explanation for the highly variable responses to mechanical loading reported in the tendon healing literature and may be useful in guiding the design of future experiments and interventions.
• Chen, K., Vigliotti, A., Bacca, M., McMeeking, R. M., Deshpande, V. S., & Holmes, J. W. (2018). Role of boundary conditions in determining cell alignment in response to stretch. Proceedings of the National Academy of Sciences of the United States of America, 115(5), 986-991.
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  The ability of cells to orient in response to mechanical stimuli is essential to embryonic development, cell migration, mechanotransduction, and other critical physiologic functions in a range of organs. Endothelial cells, fibroblasts, mesenchymal stem cells, and osteoblasts all orient perpendicular to an applied cyclic stretch when plated on stretchable elastic substrates, suggesting a common underlying mechanism. However, many of these same cells orient parallel to stretch in vivo and in 3D culture, and a compelling explanation for the different orientation responses in 2D and 3D has remained elusive. Here, we conducted a series of experiments designed specifically to test the hypothesis that differences in strains transverse to the primary loading direction give rise to the different alignment patterns observed in 2D and 3D cyclic stretch experiments ("strain avoidance"). We found that, in static or low-frequency stretch conditions, cell alignment in fibroblast-populated collagen gels correlated with the presence or absence of a restraining boundary condition rather than with compaction strains. Cyclic stretch could induce perpendicular alignment in 3D culture but only at frequencies an order of magnitude greater than reported to induce perpendicular alignment in 2D. We modified a published model of stress fiber dynamics and were able to reproduce our experimental findings across all conditions tested as well as published data from 2D cyclic stretch experiments. These experimental and model results suggest an explanation for the apparently contradictory alignment responses of cells subjected to cyclic stretch on 2D membranes and in 3D gels.

Poster Presentations

• Chen, K. (2023). Cancer and Lymphatic marker FOXC2 drives wound healing and fibrotic tissue formation.. 9th International Congress on Cancer Metastasis.