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Abstract Ionizing radiation has been pivotal in cancer therapy since its discovery. Despite its therapeutic benefits, IR causes significant acute and chronic complications due to DNA damage and the generation of reactive oxygen species, which harm nucleic acids, lipids, and proteins. While cancer cells are more vulnerable to ionizing radiation due to their inefficiency in repairing damage, healthy cells in the irradiated area also suffer. Various types of cell death occur, including apoptosis, necrosis, pyroptosis, autophagy-dependent cell death, immunogenic cell death, and ferroptosis. Ferroptosis, driven by iron-dependent lipid peroxide accumulation, has been recognized as crucial in radiation therapy’s therapeutic effects and complications, with extensive research across various tissues. This review aims to summarize the pathways involved in radiation-related ferroptosis, findings in different organs, and drugs targeting ferroptosis to mitigate its harmful effects.

Abstract Background Radiation-induced fibrosis (RIF) is an important late complication of radiation therapy, and the resulting damaging effects of RIF can significantly impact reconstructive outcomes. There is currently a paucity of effective treatment options available, likely due to the continuing knowledge gap surrounding the cellular mechanisms involved. In this study, detailed analyses of irradiated and non-irradiated human skin samples were performed incorporating histological and single-cell transcriptional analysis to identify novel features guiding development of skin fibrosis following radiation injury. Methods Paired irradiated and contralateral non-irradiated skin samples were obtained from six female patients undergoing post-oncologic breast reconstruction. Skin samples underwent histological evaluation, immunohistochemistry, and biomechanical testing. Single-cell RNA sequencing was performed using the 10X single cell platform. Cells were separated into clusters using Seurat in R. The SingleR classifier was applied to ascribe cell type identities to each cluster. Differentially expressed genes characteristic to each cluster were then determined using non-parametric testing. Results Comparing irradiated and non-irradiated skin, epidermal atrophy, dermal thickening, and evidence of thick, disorganized collagen deposition within the extracellular matrix of irradiated skin were readily appreciated on histology. These histologic features were associated with stiffness that was higher in irradiated skin. Single-cell RNA sequencing revealed six predominant cell types. Focusing on fibroblasts/stromal lineage cells, five distinct transcriptional clusters (Clusters 0–4) were identified. Interestingly, while all clusters were noted to express Cav1, Cluster 2 was the only one to also express Cav2. Immunohistochemistry demonstrated increased expression of Cav2 in irradiated skin, whereas Cav1 was more readily identified in non-irradiated skin, suggesting Cav1 and Cav2 may act antagonistically to modulate fibrotic cellular responses. Conclusion In response to radiation therapy, specific changes to fibroblast subpopulations and enhanced Cav2 expression may contribute to fibrosis. Altogether, this study introduces a novel pathway of caveolin involvement which may contribute to fibrotic development following radiation injury.

Abstract Local skin flaps are frequently employed for wound closure to address surgical, traumatic, congenital, or oncologic defects. (1) Despite their clinical utility, skin flaps may fail due to inadequate perfusion, ischemia/reperfusion injury (IRI), excessive cell death, and associated inflammatory response. (2) All of these factors contribute to skin flap necrosis in 10–15% of cases and represent a significant surgical challenge. (3, 4) Once flap necrosis occurs, it may require additional surgeries to remove the entire flap or repair the damage and secondary treatments for infection and disfiguration, which can be costly and painful. (5) In addition to employing appropriate surgical techniques and identifying healthy, well-vascularized tissue to mitigate the occurrence of these complications, there is growing interest in exploring cell-based and pharmacologic augmentation options. (6) These agents typically focus on preventing thrombosis and increasing vasodilation and angiogenesis while reducing inflammation and oxidative stress. Agents that modulate cell death pathways such as apoptosis and autophagy have also been investigated. (7) Implementation of drugs and cell lines with potentially beneficial properties have been proposed through various delivery techniques including systemic treatment, direct wound bed or flap injection, and topical application. This review summarizes pharmacologic- and cell-based interventions to augment skin flap viability in animal models, and discusses both translatability challenges facing these therapies and future directions in the field of skin flap augmentation.

Foreign body response (FBR) is a universal reaction to implanted biomaterial that can affect the function and longevity of the implant. A few studies have attempted to identify targets for treating FBR through the use of single-cell RNA sequencing (scRNA-seq), though the generalizability of these findings from an individual study may be limited. In our study, we perform a meta-analysis of scRNA-seq data from all available FBR mouse studies and integrate these data to identify gene signatures specific to FBR across different models and anatomic locations. We identify subclusters of fibroblasts and macrophages that emerge in response to foreign bodies and characterize their signaling pathways, gene ontology terms, and downstream mediators. The fibroblast subpopulations enriched in the setting of FBR demonstrated significant signaling interactions in the transforming growth factor-beta (TGF-β) signaling pathway, with known pro-fibrotic mediators identified as top expressed genes in these FBR-derived fibroblasts. In contrast, FBR-enriched macrophage subclusters highly expressed pro-fibrotic and pro-inflammatory mediators downstream of tumor necrosis factor (TNF) signaling. Cell–cell interactions were additionally interrogated using CellChat, with identification of key signaling interactions enriched between fibroblasts and macrophages in FBR. By combining multiple FBR datasets, our meta-analysis study identifies common cell-specific gene signatures enriched in foreign body reactions, providing potential therapeutic targets for patients requiring medical implants across a myriad of devices and indications.

Background:. As visibility of the transgender patient population and utilization of online resources increases, it is imperative that web-based gender-affirming surgery (GAS) materials for patients are readable, accessible, and of high quality. Methods:. A search trends analysis was performed to determine frequency of GAS-related searches over time. The top 100 most common results for GAS-related terms were analyzed using six readability formulas. Accessibility of patient-facing GAS sources was determined by categorizing types of search results. Frequency of article types was compared in low- and high-population dense areas. Quality was assigned to GAS web-based sources using the DISCERN score. Results:. Search engine trend data demonstrates increasing occurrence of searches related to GAS. Readability scores of the top 100 online sources for GAS were discovered to exceed recommended levels for patient proficiency. Availability of patient-facing online information related to GAS was found to be 60%, followed by information provided by insurance companies (17%). Differences in availability of online resources in varying dense cities were found to be minimal. The average quality of sources determined by the DISCERN score was found to be 3, indicating “potential important shortcomings.” Conclusions:. Despite increasing demand for web-based GAS information, the readability of online resources related to GAS was found to be significantly greater than the grade level of proficiency recommended for patients. A high number of nonpatient-facing search results appear in response to GAS search terms. Quality sources are still difficult for patients to find, as search results have a high incidence of low-quality resources.

Abstract 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.

Abstract Background Radiation-induced skin injury is a well-known risk factor for impaired wound healing. Over time, the deleterious effects of radiation on skin produce a fibrotic, hypovascular dermis poorly suited to wound healing. Despite increasing understanding of the underlying pathophysiology, therapeutic options remain elusive. Deferoxamine (DFO), an iron-chelating drug, has been shown in prior murine studies to ameliorate radiation-induced skin injury as well as improve wound healing outcomes in various pathologic conditions when administered transdermally. In this preclinical study, we evaluated the effects of deferoxamine on wound healing outcomes in chronically irradiated murine skin. Methods Wild-type mice received 30 Gy of irradiation to their dorsal skin and were left to develop chronic fibrosis. Stented excisional wounds were created on their dorsal skin. Wound healing outcomes were compared across 4 experimental conditions: DFO patch treatment, vehicle-only patch treatment, untreated irradiated wound, and untreated nonirradiated wounds. Gross closure rate, wound perfusion, scar elasticity, histology, and nitric oxide assays were compared across the conditions. Results Relative to vehicle and untreated irradiated wounds, DFO accelerated wound closure and reduced the frequency of healing failure in irradiated wounds. DFO augmented wound perfusion throughout healing and upregulated angiogenesis to levels observed in nonirradiated wounds. Histology revealed DFO increased wound thickness, collagen density, and improved collagen fiber organization to more closely resemble nonirradiated wounds, likely contributing to the observed improved scar elasticity. Lastly, DFO upregulated inducible nitric oxide synthase and increased nitric oxide production in early healing wounds. Conclusion Deferoxamine treatment presents a potential therapeutic avenue through which to target impaired wound healing in patients following radiotherapy.

Wound healing results in the formation of scar tissue which can be associated with functional impairment, psychological stress, and significant socioeconomic cost which exceeds 20 billion dollars annually in the United States alone. Pathologic scarring is often associated with exaggerated action of fibroblasts and subsequent excessive accumulation of extracellular matrix proteins which results in fibrotic thickening of the dermis. In skin wounds, fibroblasts transition to myofibroblasts which contract the wound and contribute to remodeling of the extracellular matrix. Mechanical stress on wounds has long been clinically observed to result in increased pathologic scar formation, and studies over the past decade have begun to uncover the cellular mechanisms that underly this phenomenon. In this article, we will review the investigations which have identified proteins involved in mechano-sensing, such as focal adhesion kinase, as well as other important pathway components that relay the transcriptional effects of mechanical forces, such as RhoA/ROCK, the hippo pathway, YAP/TAZ, and Piezo1. Additionally, we will discuss findings in animal models which show the inhibition of these pathways to promote wound healing, reduce contracture, mitigate scar formation, and restore normal extracellular matrix architecture. Recent advances in single cell RNA sequencing and spatial transcriptomics and the resulting ability to further characterize mechanoresponsive fibroblast subpopulations and the genes that define them will be summarized. Given the importance of mechanical signaling in scar formation, several clinical treatments focused on reducing tension on the wound have been developed and are described here. Finally, we will look toward future research which may reveal novel cellular pathways and deepen our understanding of the pathogenesis of pathologic scarring. The past decade of scientific inquiry has drawn many lines connecting these cellular mechanisms that may lead to a map for the development of transitional treatments for patients on the path to scarless healing.

Summary: 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),1 Galiano et al. (2004),2 and Mascharak et al. (2022).3 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Background:. Distraction osteogenesis (DO) is used for skeletal defects; however, up to 50% of cases exhibit complications. Previous mouse models of long bone DO have been anecdotally hampered by postoperative complications, expense, and availability. To improve clinical techniques, cost-effective, reliable animal models are needed. Our focus was to develop a new mouse tibial distractor, hypothesized to result in successful, complication-free DO. Methods:. A lightweight tibial distractor was developed using CAD and 3D printing. The device was fixed to the tibia of C57Bl/6J mice prior to osteotomy. Postoperatively, mice underwent 5 days latency, 10 days distraction (0.15 mm every 12 hours), and 28 days consolidation. Bone regeneration was examined on postoperative day 43 using micro-computed tomography (μCT) and Movat’s modified pentachrome staining on histology (mineralized volume fraction and pixels, respectively). Costs were recorded. We compared cohorts of 11 mice undergoing sham, DO, or acute lengthening (distractor acutely lengthened 3.0 mm). Results:. The histological bone regenerate was significantly increased in DO (1,879,257 ± 155,415 pixels) compared to acute lengthening (32847 ± 1589 pixels) (P < 0.0001). The mineralized volume fraction (bone/total tissue volume) of the regenerate was significantly increased in DO (0.9 ± 0.1) compared to acute lengthening (0.7 ± 0.1) (P < 0.001). There was no significant difference in bone regenerate between DO and sham. The distractor was relatively low cost ($11), with no complications. Conclusions:. Histology and µCT analysis confirmed that the proposed tibial DO model resulted in successful bone formation. Our model is cost-effective and reproducible, enabling implementation in genetically dissectible transgenic mice.

There is undisputable benefit in translating basic science research concretely into clinical practice, and yet, the vast majority of therapies and treatments fail to achieve approval. The rift between basic research and approved treatment continues to grow, and in cases where a drug is granted approval, the average time from initiation of human trials to regulatory marketing authorization spans almost a decade. Albeit with these hurdles, recent research with deferoxamine (DFO) bodes significant promise as a potential treatment for chronic, radiation-induced soft tissue injury. DFO was originally approved by the Food and Drug Administration (FDA) in 1968 for the treatment of iron overload. However, investigators more recently have posited that its angiogenic and antioxidant properties could be beneficial in treating the hypovascular and reactive-oxygen species-rich tissues seen in chronic wounds and radiation-induced fibrosis (RIF). Small animal experiments of various chronic wound and RIF models confirmed that treatment with DFO improved blood flow and collagen ultrastructure. With a well-established safety profile, and now a strong foundation of basic scientific research that supports its potential use in chronic wounds and RIF, we believe that the next steps required for DFO to achieve FDA marketing approval will include large animal studies and, if those prove successful, human clinical trials. Though these milestones remain, the extensive research thus far leaves hope for DFO to bridge the gap between bench and wound clinic in the near future.

Historically believed to be a homogeneous cell type that is often overlooked, fibroblasts are more and more understood to be heterogeneous in nature. Though the mechanisms behind how fibroblasts participate in homeostasis and pathology are just beginning to be understood, these cells are believed to be highly dynamic and play key roles in fibrosis and remodeling. Focusing primarily on fibroblasts within the skin and during wound healing, we describe the field’s current understanding of fibroblast heterogeneity in form and function. From differences due to embryonic origins to anatomical variations, we explore the diverse contributions that fibroblasts have in fibrosis and plasticity. Following this, we describe molecular techniques used in the field to provide deeper insights into subpopulations of fibroblasts and their varied roles in complex processes such as wound healing. Limitations to current work are also discussed, with a focus on future directions that investigators are recommended to take in order to gain a deeper understanding of fibroblast biology and to develop potential targets for translational applications in a clinical setting.

Pancreatic cancer is one of the deadliest forms of cancer with one of the lowest 5-year survival rates of all cancer types. A defining characteristic of pancreatic cancer is the existence of dense desmoplastic stroma that, when exposed to stimuli such as cytokines, growth factors, and chemokines, generate a tumor-promoting environment. Cancer-associated fibroblasts (CAFs) are activated during the progression of pancreatic cancer and are a crucial component of the tumor microenvironment (TME). CAFs are primarily pro-tumorigenic in their activated state and function as promoters of cancer invasion, proliferation, metastasis, and immune modulation. Aided by many signaling pathways, cytokines, and chemokines in the tumor microenvironment, CAFs can originate from many cell types including resident fibroblasts, mesenchymal stem cells, pancreatic stellate cells, adipocytes, epithelial cells, endothelial cells, and other cell types. CAFs are a highly heterogeneous cell type expressing a variety of surface markers and performing a wide range of tumor promoting and inhibiting functions. Single-cell transcriptomic analyses have revealed a high degree of specialization among CAFs. Some examples of CAF subpopulations include myofibrotic CAFs (myCAFs), which exhibit a matrix-producing contractile phenotype; inflammatory CAFs (iCAF) that are classified by their immunomodulating, secretory phenotype; and antigen-presenting CAFs (apCAFs), which have antigen-presenting capabilities and express Major Histocompatibility Complex II (MHC II). Over the last several years, various attempts have been undertaken to describe the mechanisms of CAF–tumor cell interaction, as well as CAF–immune cell interaction, that contribute to tumor proliferation, invasion, and metastasis. Although our understanding of CAF biology in cancer has steadily increased, the extent of CAFs heterogeneity and their role in the pathobiology of pancreatic cancer remains elusive. In this regard, it becomes increasingly evident that further research on CAFs in pancreatic cancer is necessary.

Cranial sutures are major growth centers for the skull vault and premature fusion leads to pathological fusion, craniosynostosis. Here the authors isolate Wnt responsive skeletal stem and progenitor cells from sutures, that can be transplanted together with Wnt3a protein to repair craniosynostosis in a mouse model.

Abstract Background Older cancer survivors have high rates of long‐term opioid therapy (≥90 days/year). However, the geographical and temporal variation in long‐term opioid therapy rates for older cancer survivors is not known. Methods A retrospective cohort study was conducted using SEER‐Medicare data. Persons aged ≥66 years, diagnosed with breast, colorectal, lung, or prostate cancer from 1991 to 2011, and alive ≥5 years after diagnosis were included. Persons were followed from 1/1/2008 until 12/31/2016. Persons were assigned to a census region in their state of residence each year. Individuals who were covered by an opioid prescription for at least 90 days in a calendar year were classified as having received long‐term opioid therapy. Multivariable analysis was conducted using generalized estimating equations. Results Temporal trends significantly varied by region (p

Abstract Fat grafting can reduce radiation‐induced fibrosis. Improved outcomes are found when fat grafts are enriched with adipose‐derived stromal cells (ASCs), implicating ASCs as key drivers of soft tissue regeneration. We have identified a subpopulation of ASCs positive for CD74 with enhanced antifibrotic effects. Compared to CD74− and unsorted (US) ASCs, CD74+ ASCs have increased expression of hepatocyte growth factor, fibroblast growth factor 2, and transforming growth factor β3 (TGF‐β3) and decreased levels of TGF‐β1. Dermal fibroblasts incubated with conditioned media from CD74+ ASCs produced less collagen upon stimulation, compared to fibroblasts incubated with media from CD74− or US ASCs. Upon transplantation, fat grafts enriched with CD74+ ASCs reduced the stiffness, dermal thickness, and collagen content of overlying skin, and decreased the relative proportions of more fibrotic dermal fibroblasts. Improvements in several extracellular matrix components were also appreciated on immunofluorescent staining. Together these findings indicate CD74+ ASCs have antifibrotic qualities and may play an important role in future strategies to address fibrotic remodeling following radiation‐induced fibrosis.

Abstract Fat grafting is a surgical technique able to reconstruct and regenerate soft tissue. The adipose‐derived stromal cells (ASCs) within the stromal vascular fraction are believed to drive these beneficial effects. ASCs are increasingly recognized to be a heterogeneous group, comprised of multiple stem and progenitor subpopulations with distinct functions. We hypothesized the existence of an ASC subpopulation with enhanced angiogenic potential. Human ASCs that were CD34+CD146+, CD34+CD146−, or CD34+ unfractionated (UF) were isolated by flow cytometry for comparison of expression of proangiogenic factors and endothelial tube‐forming potential. Next, lipoaspirate was enriched with either CD34+CD146+, CD34+CD146−, CD34+ UF ASCs, or was not enriched, and grafted beneath the scalp skin of immunodeficient CD‐1 Nude mice (10 000 cells/200 μL/graft). Fat retention was monitored radiographically more than 8 weeks and fat grafts were harvested for histological assessment of quality and vascularization. The CD34+CD146+ subpopulation comprised ~30% of ASCs, and exhibited increased expression of vascular endothelial growth factor and angiopoietin‐1 compared to CD34+CD146− and CD34+ UF ASCs, and increased expression of fibroblast growth factor‐2 compared to CD34+CD146− ASCs. The CD34+CD146+ subpopulation exhibited enhanced induction of tube‐formation compared to CD34+CD146− ASCs. Upon transplantation, fat enriched CD34+CD146+ ASCs underwent less resorption and had improved histologic quality and vascularization. We have identified a subpopulation of CD34+ ASCs with enhanced angiogenic effects in vitro and in vivo, likely mediated by increased expression of potent proangiogenic factors. These findings suggest that enriching lipoaspirate with CD34+CD146+ ASCs may enhance fat graft vascularization and retention in the clinical setting.

Salivary gland hypofunction causes significant morbidity and loss of quality of life for head and neck cancer patients treated with radiotherapy. Preventing hypofunction is an unmet therapeutic need. We used an adeno-associated virus serotype 2 (AAV2) vector expressing the human neurotrophic factor neurturin (CERE-120) to treat murine submandibular glands either pre- or post-irradiation (IR). Treatment with CERE-120 pre-IR, not post-IR, prevented hypofunction. RNA sequencing (RNA-seq) analysis showed reduced gene expression associated with fibrosis and the innate and humoral immune responses. We then used a minipig model with CERE-120 treatment pre-IR and also compared outcomes of the contralateral non-IR gland. Analysis of gene expression, morphology, and immunostaining showed reduced IR-related immune responses and improved secretory mechanisms. CERE-120 prevented IR-induced hypofunction and restored immune homeostasis, and there was a coordinated contralateral gland response to either damage or treatment. CERE-120 gene therapy is a potential treatment for head and neck cancer patients to influence communication among neuronal, immune, and epithelial cells to prevent IR-induced salivary hypofunction and restore immune homeostasis.

Previously, using plankton tows, and emergence and settlement traps, we documented persistent widespread nocturnal emergence, and planktonic redistribution, of benthic macroinvertebrates along the coral reef–seagrass interface at two geographically separated locations. We also documented that emergence intensity varies with distance from the reef, leading us to hypothesize that the spatial pattern of emergence is determined by the foraging patterns of nocturnally active, bottom-feeding, mid-level consumers (mainly grunts). In this second study, we coupled those previously published data with nocturnal fish surveys concurrently conducted along belt transects placed at the same locations as the emergence trap collections, and a controlled laboratory experiment, to test this hypothesis. The results of these analyses find that variability in the density of nocturnally active, bottom-feeding fish is strongly positively correlated with emergence intensity, regardless of site or season. Results from the laboratory experiments show that nocturnal invertebrate emergence is significantly higher in the presence of one bottom-feeding fish (the blue-striped grunt Haemulon sciurus) than in microcosms that do not contain this predator. Overall, this study shows that such processes may explain how benthic prey can avoid capture by nocturnally active, bottom-feeding predators and persist in the predator-rich seagrass habitats that surround coral reefs in the Florida Keys National Marine Sanctuary. This study also points out the need to consider nocturnal processes when studying seagrass biodiversity in a predator-rich environment.

Royal jelly is the queen-maker for the honey bee that also has effects on longevity, fertility, and regeneration in mammals. Here the authors provide evidence that its major protein component Royalactin, and the mammalian structural analog Regina, maintain pluripotency in mouse ESCs by activating a ground-state pluripotency-like gene network.

In the fields of oral and craniomaxillofacial surgery, regeneration of multiple tissue types—including bone, skin, teeth, and mucosal soft tissue—is often a desired outcome. However, limited endogenous capacity for regeneration, as well as predisposition of many tissues to fibrotic healing, may prevent recovery of normal form and function for patients. Recent basic science research has advanced our understanding of molecular and cellular pathways of repair in the oral/craniofacial region and how these are influenced by local microenvironment and embryonic origin. Here, we review the current state of knowledge in oral and craniomaxillofacial tissue repair/regeneration in four key areas: bone (in the context of calvarial defects and mandibular regeneration during distraction osteogenesis); skin (in the context of cleft lip/palate surgery); oral mucosa (in the context of minimally scarring repair of mucosal injuries); and teeth (in the context of dental disease/decay). These represent four distinct healing processes and outcomes. We will discuss both divergent and conserved pathways of repair in these contexts, with an eye toward fundamental mechanisms of regeneration vs. fibrosis as well as translational research directions. Ultimately, this knowledge can be leveraged to develop new cell-based and molecular treatment strategies to encourage bone and soft tissue regeneration in oral and craniomaxillofacial surgery.

The infection of surgically placed implants is a problem that is both large in magnitude and that broadly affects nearly all surgical specialties. Implant-associated infections deleteriously affect patient quality-of-life and can lead to greater morbidity, mortality, and cost to the health care system. The impact of this problem has prompted extensive pre-clinical and clinical investigation into decreasing implant infection rates. More recently, antimicrobial approaches that modify or treat the implant directly have been of great interest. These approaches include antibacterial implant coatings (antifouling materials, antibiotics, metal ions, and antimicrobial peptides), antibacterial nanostructured implant surfaces, and antibiotic-releasing implants. This review provides a compendium of these approaches and the clinical applications and outcomes. In general, implant-specific modalities for reducing infections have been effective; however, most applications remain in the preclinical or early clinical stages.

Purpose: Scarring in the mouse dorsal dermis is mediated by pro-fibrotic, Engrailed-1 lineage-positive fibroblasts (EPFs). We recently showed that mechanotransduction blockade (YAP inhibition, using the drug verteporfin), results in complete wound regeneration, with full recovery of normal dermal appendages (hair follicles, glands), extracellular matrix (ECM) architecture, and tensile strength. This regenerative outcome following verteporfin treatment is mediated by Engrailed-1 lineage-negative fibroblasts (ENFs). The complex milieu of cell types and molecular signals involved in wound repair makes it difficult to study using any single data modality. Thus, we sought to use a holistic approach, incorporating multiple high-throughput, high-dimensional analyses, to define the divergent molecular events distinguishing typical scarring healing from verteporfin-induced wound regeneration. Methods: C57BL/6J mice underwent dorsal splinted excisional wounding per standard protocol. Wounds were treated with local injection of either verteporfin or vehicle control (PBS) on POD 0. We harvested unwounded skin and wounds at POD 2, 7, 14, and 30 (n=5 mice/timepoint and treatment) and subjected wound cells to three analyses: single-cell RNA-sequencing (scRNA-seq, using 10X Genomics Chromium); timsTOF, a recently-developed, high-throughput proteomic sequencing platform; and a novel machine learning algorithm for quantitatively comparing ECM ultrastructure. Results: Pseudotime analysis (Monocle3) of pooled scRNA-seq data revealed that fibroblasts followed two distinct transcriptional trajectories, one characterized by mechanical activation (En-1 lineage-positive, “fibrotic” trajectory) and the other characterized by developmental and regenerative pathways (En-1 lineage-negative; Rspo1, Dkk2/3, Trps1). Cross-platform data integration confirmed that fibroblasts in the fibrotic trajectory correlated with myofibroblast proteomic signatures (Col1a1/2, Fn1, etc.) and fibrotic/scar ECM features. In contrast, fibroblasts in the regenerative trajectory negatively correlated with myofibroblast markers and were associated with a “basket-weave” ECM pattern quantitatively indistinguishable from that of unwounded skin. Our integrated dataset suggested an important role for Wnt pathway proteins in ENF-mediated skin regeneration, so we compared POD 14 scars and regenerated wounds by multiplexed in situ hybridization (RNAScope) for Rspo1 (Wnt agonist), Trps1 (master hair follicle regulator), Ank1 (YAP target gene), and Dpp4 (EPF marker). Quantification of RNA granules across thousands of cells using a custom image analysis pipeline revealed that ENF-mediated healing (low Dpp4) in YAP-inhibited (low Ank1) wounds yielded regeneration of functional hair follicles through Wnt-mediated pathway activation (high Rpos1, Trps1). These data suggest that YAP inhibition unlocks wound regeneration via Wnt-active, En-1 lineage-negative fibroblasts. Conclusion: By studying regenerating (verteporfin-treated) versus scarring wounds across multiple healing timepoints and high-dimensional data modalities, we were able to profile fibrotic versus regenerative healing at unprecedented depth. Our integrated analysis revealed that dermal fibroblasts in these two wound settings exhibit distinct molecular trajectories defined by divergent transcriptomic, proteomic, and ultrastructural properties. Further, we found that wound regeneration in the context of verteporfin treatment is associated with suppression of mechanical signaling and activation of key Wnt pathway members including Trps1 (a gene with known hair follicle developmental roles). These results could have important implications for both the fundamental study of wound healing and potential anti-scarring therapeutic avenues

Cell therapy for treatment of peripheral arterial disease (PAD) is a promising approach but is limited by poor cell survival when cells are delivered using saline. The objective of this study was to examine the feasibility of aligned nanofibrillar scaffolds as a vehicle for the delivery of human stromal vascular fraction (SVF), and then to assess the efficacy of the cell-seeded scaffolds in a murine model of PAD. Flow cytometric analysis was performed to characterize the phenotype of SVF cells from freshly isolated lipoaspirate, as well as after attachment onto aligned nanofibrillar scaffolds. Flow cytometry results demonstrated that the SVF consisted of 33.1 ± 9.6% CD45+ cells, a small fraction of CD45–/CD31+ (4.5 ± 3.1%) and 45.4 ± 20.0% of CD45–/CD31–/CD34+ cells. Although the subpopulations of SVF did not change significantly after attachment to the aligned nanofibrillar scaffolds, protein secretion of vascular endothelial growth factor (VEGF) significantly increased by six-fold, compared to SVF cultured in suspension. Importantly, when SVF-seeded scaffolds were transplanted into immunodeficient mice with induced hindlimb ischemia, the cell-seeded scaffolds induced a significant higher mean perfusion ratio after 14 days, compared to cells delivered using saline. Together, these results show that aligned nanofibrillar scaffolds promoted cellular attachment, enhanced the secretion of VEGF from attached SVF cells, and their implantation with attached SVF cells stimulated blood perfusion recovery. These findings have important therapeutic implications for the treatment of PAD using SVF.

Summary:. Fibroblasts’ integral role in tissue development, maintenance, and disease represents a fast-growing field of basic science research. Although fibroblasts were long thought to be a homogeneous cell population, recent research has illuminated the unforeseen complexity of these cells, giving rise to the rapidly expanding research field of “fibroblast heterogeneity.” Fibroblasts play a critical role in states of tissue fibrosis such as skin scarring, which affects hundreds of millions of patients annually and causes severe aesthetic, developmental, and functional morbidity. Beyond scarring, major organ fibrosis is an enormous public health concern responsible for nearly half of all deaths in the United States. Because fibrosis is a conserved response to tissue damage in all organs, the study of fibroblasts throughout the body may help us to understand their role in the conditions most relevant to plastic and reconstructive surgery—for instance, skin scarring (eg, from burns, traumatic lacerations, or surgical incisions), “pathological” scarring (hypertrophic scars, keloids), and capsular contracture. Here, we present a basic science review of fibroblast heterogeneity in wound healing, cancer, organ fibrosis, and human dermal architecture. The field of fibroblast heterogeneity is young, and many of the insights discussed have yet to be translated clinically. However, plastic surgeons stand in a unique position to bridge these discoveries into clinical realities. We hope this information can spur readers to consider both what questions in plastic surgery can be studied from the lens of fibroblast heterogeneity, and how these preclinical insights can be translated to improving care of our patients.

Background:. Abdominoperineal resection (APR) and pelvic exenteration continue to be common procedures for the treatment of colorectal malignancy. The workhorse flap for reconstruction in these instances has been the vertical rectus abdominis myocutaneous flap. The associated donor site morbidity, however, cannot be ignored. Here, we provide a review of the literature and present the senior author’s (A.M.) experience using the pedicled anterolateral thigh (ALT) flap for reconstruction of soft tissue defects following APR and pelvic exenteration. Methods:. Patients who underwent pelvic/perineal reconstruction with pedicled ALT flaps between 2017 and 2019 were included in the study. Parameters of interest included age, gender, body mass index, comorbidities, history of radiation, extent of ablative surgery, and postoperative complication rate. Results:. A total of 23 patients (16 men and 7 women) with a median age and body mass index of 66 years (inter-quartile range [IQR]: 49–71 years) and 24.9 kg/m2 (IQR: 24.2–26.7 kg/m2) were included in the study, respectively. Thirteen (56.5%) patients presented with rectal cancer, 5 (21.7%) with anal squamous cell carcinoma (SCC), 4 (17.4%) with Crohn’s disease, and 1 (4.3%) with Paget’s disease. Nineteen patients (82.6%) received neoadjuvant radiation. Nine (39.1%) patients experienced 11 complications (2 major and 9 minor). The most common complication was partial perineal wound dehiscence (N = 6 [26.1%]). Stable soft tissue coverage was achieved in all but one patient. Conclusions:. The ALT flap allows for stable soft tissue coverage following APR and pelvic exenteration without being associated with abdominal donor site morbidity. Consideration to its use as a first-line reconstructive option should be given in pelvic/perineal reconstruction.

Summary: Organoids are in vitro miniaturized organ models—or, colloquially, “organs in a dish.” These 3-dimensional, multicellular structures are classically derived from pluripotent or multipotent stem cells. When guided by tissue-specific molecular factors, these cells exhibit self-organizing abilities that allow them to accurately recapitulate the architecture and function of the organ of interest. Organoid technology is a rapidly expanding field that endows researchers with an unprecedented ability to recreate, study, and manipulate complex biologic processes in vitro. When compared with standard 2- and 3-dimensional culture systems, which rely on co-culturing pre-established cell types, organoids provide a more biomimetic model with which to study the intercellular interactions necessary for in vivo organ function and architecture. Organoids have the potential to impact all avenues of medicine, including those fields most relevant to plastic and reconstructive surgery such as wound healing, oncology, craniofacial reconstruction, and burn care. In addition to their ability to serve as a novel tool for studying human-specific disease, organoids may be used for tissue engineering with the goal of developing biomimetic soft-tissue substitutes, which would be especially valuable to the plastic surgeon. Although organoids hold great promise for the field of plastic surgery, technical challenges in creating vascularized, multilineage organoids must be overcome to allow for the integration of this technology in clinical practice. This review provides a brief history of the organoid, highlights its potential clinical applications, discusses certain limitations, and examines the impact that this technology may have on the field of plastic and reconstructive surgery.

Targeted genetic dissection of tissues can be used to identify cell populations and lineages. Here the authors develop 4-hydroxytamoxifen liposomes for the localized induction of CreERT2.

Purpose: Fat grafting is known to rejuvenate and improve fibrosis in irradiated skin, but the underlying mechanisms behind these effects are poorly understood. We have previously identified the Engrailed-1 fibroblast sub-population as having a role in post-natal dorsal skin fibrosis in mice. Using a radiation model to the scalp of Engrailed-1 mice, we sought to better understand the regenerative effects of fat grafting in irradiated tissue. Methods: Adult (60-day old) En1Cre;R26mTmG transgenic mice (n=5) underwent total body irradiation with 9 Gy for immunodepletion. Mice were then immediately reconstituted with 2 million nucleated bone marrow cells from donor NSG(NOD.CB17-Prkdcsscid/J) mice via retro-orbital injections. Reconstitution was confirmed with fluorescence-activated cell sorting (FACS) 2 weeks after whole body irradiation. Mice scalps were then irradiated with 5 Gy every other day for 12 days (30 Gy total) and allowed to recover for 4 weeks to facilitate fibrotic conversion. Irradiated scalps were grafted with 200μL of fresh human lipoaspirate. Graph retention was measured in-vivo for 8 weeks using Micro-Computed Tomography Scanner (Bruker SkyScan 1726™) and scalp skin was harvest for histology. Results: Two weeks post bone-marrow transplant, >90% of circulating hematopoietic cells (CD45+) cells in En1Cre;R26mTmG reporter mice were non-fluorescent, signifying successful reconstitution. Volumetric analysis of fat grafting in-vivo was performed using 3-dimensional reconstruction. At 8 weeks post-grafting, 4 of the 5 grafts demonstrated >50% graft retention, confirming successful grafting. Histological sections of scalp skin 8 weeks post-grafting demonstrated significantly less En1+GFP cells compared to non-grafted scalp skin, signifying less presence of En1+ fibroblasts. Furthermore, histology demonstrated significantly less dermal thickening, epidermal thinning, and collagen deposition and disorganization in fat grafted irradiated scalp skin compared to non-grafted scalp skin. Conclusion: Fat grafting mitigates radiation-induced fibrosis in scalp skin by decreasing collagen deposition, remodeling collagen formation, decreasing epidermal thinning, and reducing presence of pro-fibrotic fibroblast sub-populations.