Faculty Bibliography

PURPOSE/OBJECTIVE:The various physiological profiles comprising vascularized composite allografts (VCAs) pose unique challenges to preservation. Minimizing ischemia, reperfusion injury, and rejection remains a primary focus of graft pretreatments (PTs). Currently, the gold standard PT consists of flushing the graft and placing it in static cold storage in the University of Wisconsin solution. With this method, graft viability is limited to 4 to 6 hours. Prolonging this time limit will increase donor allocation radius, access to care, and positive patient outcomes. We aimed to evaluate novel PTs that could potentially enhance and lengthen VCA viability. METHODS:Following PRISMA (Preferred Reporting Items for Systematic reviews and Meta-Analyses) guidelines, we conducted a comprehensive literature search of EMBASE, Cochrane, and PubMed. Studies had to be published prior to June 15, 2022. PTs had to target cell physiology, rather than immunogenicity. We extracted data including study design, PT details, evaluation metrics, and outcomes. RESULTS:We identified 13 studies, categorized into 3 groups: solution-based alterations to the gold standard, ex vivo perfusion, and other novel techniques. The incorporation of hydrogen sulfide and Perfadex as solutions in the gold standard protocol demonstrated a 6-day delay in rejection and limited reperfusion injury markers, respectively. In an ex vivo perfusion study, after 24 hours of PT and 12 hours posttransplant, VCA muscle contractility remained close to normal. The gold standard PT did not demonstrate the same success. However, graft weight gain, up to 50% of baseline among the reviewed articles, is a prominent adverse effect of perfusion. Another technique, cryopreservation, displayed 90% graft failure by venous thrombosis, despite high free graft viability following 2 weeks of storage. CONCLUSIONS:This study of PT modalities found a variety of encouraging preservation techniques for grafts with high levels of tissue diversity. Ex vivo perfusion dominated PT innovation with promising results in preserving the viability and functionality of muscle, which is central to the restoration of movement. Future studies are necessary to evaluate long-term graft outcomes and to optimize PT protocols for extended preservation times to ensure clinical relevance.

Aging populations worldwide face an increasing burden of age-related chronic conditions, necessitating a deeper understanding of the underlying mechanisms. Purine metabolism has emerged as a crucial player in the pathophysiology of aging, affecting various tissues and organs. Dysregulation of purine metabolism, particularly alterations in extracellular adenosine levels and adenosine receptor signaling, contributes to age-related musculoskeletal problems, cardiovascular diseases, inflammation, and impaired immune responses. Changes in purine metabolism are associated with diminished tissue repair and regeneration, altered bone density, and impaired muscle regeneration. Mechanistically, age-related alterations in purine metabolism involve reductions in extracellular adenosine production, impaired autocrine signaling, and dysregulated expression of CD73 and CD39. Targeting adenosine receptors, such as A_2A and A_2B receptors, emerges as a promising therapeutic approach to mitigate age-related conditions, including sarcopenia, obesity, osteoarthritis, and impaired wound healing. Since we cannot reverse time, understanding the intricate molecular interplay between purine metabolism and aging-related pathologies holds significant potential for developing novel therapeutic strategies to improve the health and quality of life of aging populations. In this review, we compile the findings related to purine metabolism during aging in several tissues and organs and provide insights into how these signals can be manipulated to circumvent the deleterious effects of the passage of time on our body.

BACKGROUND:Vascularized composite allotransplantation (VCA) holds significant promise for patients with complex structural defects, providing solutions unattainable through traditional methods. Despite technical successes, graft rejection and ischemia-reperfusion injury (IRI) present major challenges, with high rejection rates even under modern immunosuppression protocols. This review synthesizes current literature on immunologic pretreatments (IPTs) designed to mitigate these issues, focusing on interventions applied to donor tissues between procurement and transplantation. METHODS:A systematic review following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines identified 11 relevant studies, categorizing IPTs into donor tissue modification (DTM), deoxygenated perfusate, and oxygenation methods. RESULTS:DTM, the most common IPT method, shows promise in reducing immunogenicity and prolonging graft survival, primarily through techniques such as recipient bone marrow-derived cell conditioning and MHC-I knockdown using small interfering RNA (siRNA). Deoxygenated perfusate studies highlighted mitomycin C's potential in reducing immune response and extending graft viability. Oxygenation methods, aimed at minimizing IRIs, utilized perfusion techniques to maintain graft viability ex vivo. CONCLUSIONS:Although IPTs for extending graft survival have seen moderate clinical translation, those targeting immunogenicity remain largely experimental. This review underscores the potential of these IPT modalities to improve VCA outcomes by reducing rejection and IRIs. However, it also highlights the need for further research, particularly multi-center clinical trials, to validate these techniques for broader clinical adoption. This comprehensive synthesis aims to guide future studies and enhance clinical strategies for VCA, ultimately improving patient outcomes.

Hair follicle neogenesis (HFN) occurs following large skin excisions in mice, serving as a rare regenerative model in mammalian wound healing. Wound healing typically results in fibrosis in mice and humans. We previously showed small skin excisions in mice result in scarring devoid of HFN, displaying features of non-regenerative healing, and Hedgehog (Hh) activation in the dermis of such wounds can induce HFN. In this study, we sought to verify the role of dermal Wnt/β-catenin signaling in HFN, as this pathway is essential for HF development, but is also paradoxically well-characterized in fibrosis of adult wounds. By deletion of β-catenin in large wound myofibroblasts, we show Wnt/β-catenin signaling is required for endogenous mechanisms of HFN. Through utilizing a combined mouse model that simultaneously induces deletion of β-catenin and constitutive activation of Smoothened (Smo) in myofibroblasts, we also found β-catenin is required for Hh-driven DP formation. Transcriptome analysis confirms Wnt/β-catenin and Hh pathways are activated in dermal papilla (DP) cells. Our results indicate that Wnt-active fibrotic status may also create a permissive state for the regenerative function of Hh, suggesting that activation of both Wnt and Hh pathways in skin wound fibroblasts must be ensured in future strategies to promote HFN.

BACKGROUND/UNASSIGNED:A deeper understanding of acute rejection in vascularized composite allotransplantation is paramount for expanding its utility and longevity. There remains a need to develop more precise and accurate tools for diagnosis and prognosis of these allografts, as well as alternatives to traditional immunosuppressive regimens. METHODS/UNASSIGNED:Twenty-seven skin biopsies collected from 3 vascularized composite allotransplantation recipients, consisting of face and hand transplants, were evaluated by histology, immunohistochemistry staining, and gene expression profiling. RESULTS/UNASSIGNED:significantly predicted inflammation specific to vascularized composite allografts that required therapeutic intervention. CONCLUSIONS/UNASSIGNED:The mechanism of vascularized composite allograft-specific inflammation and rejection appears to be conserved across different patients and skin on different anatomical sites. A concise gene signature can be utilized to ascertain graft status along with a continuous scale, providing valuable diagnostic and prognostic information to supplement current gold standards of graft evaluation.

Exosomes are being increasingly explored in biomedical research for wound healing applications. Exosomes can improve blood circulation and endocrine signaling, resulting in enhanced cell regeneration. However, exosome treatments suffer from low retention and bioavailability of exosomes at the wound site. Hydrogels are a popular tool for drug delivery due to their ability to encapsulate drugs in their network and allow for targeted release. Recently, hydrogels have proven to be an effective method to provide increased rates of wound healing when combined with exosomes that can be applied noninvasively. We have designed a series of single-domain protein-based hydrogels capable of physical cross-linking and upper critical solution temperature (UCST) behavior. Hydrogel variant Q5, previously designed with improved UCST behavior and a significantly enhanced gelation rate, is selected as a candidate for encapsulation release of exosomes dubbed Q5Exo. Q5Exo exhibits low critical gelation times and significant decreases in wound healing times in a diabetic mouse wound model showing promise as an exosome-based drug delivery tool and for future hybrid, noninvasive protein-exosome design.

OBJECTIVE:Rejection is common and pernicious following Vascularized Composite Allotransplantation (VCA). Current monitoring and diagnostic modalities include the clinical exam which is subjective and biopsy with dermatohistopathologic Banff grading, which is subjective and invasive. We reviewed literature exploring non- and minimally invasive modalities for diagnosing and monitoring rejection (NIMMs) in VCA. METHODS:PubMed, Cochrane, and Embase databases were queried, 3125 unique articles were reviewed, yielding 26 included studies exploring 17 distinct NIMMs. Broadly, NIMMs involved Imaging, Liquid Biomarkers, Epidermal Sampling, Clinical Grading Scales, and Introduction of Additional Donor Tissue. RESULTS:Serum biomarkers including MMP3 and donor-derived microparticles rose with rejection onset. Epidermal sampling non-invasively enabled measurement of cytokine & gene expression profiles implicated in rejection. Both hold promise for monitoring. Clinical grading scales were useful diagnostically as was reflection confocal microscopy. Introducing additional donor tissue showed promise for preemptively identifying rejection but requires additional allograft tissue burden for the recipient. CONCLUSION/CONCLUSIONS:NIMMs have the potential to dramatically improve monitoring and diagnosis in VCA. Many modalities show promise however, additional research is needed and a multimodal algorithmic approach should be explored.

Osteoarthritis (OA) pathogenesis is associated with reduced chondrocyte homeostasis and increased levels of cartilage cellular senescence. Chondrosenescence is the development of cartilage senescence that increases with aging joints and disrupts chondrocyte homeostasis and is associated with OA. Adenosine A2A receptor (A2AR) activation in cartilage via intra-articular injection of liposomal A2AR agonist, liposomal-CGS21680, leads to cartilage regeneration in vivo and chondrocyte homeostasis. A2AR knockout mice develop early OA isolated chondrocytes demonstrate upregulated expression of cellular senescence and aging-associated genes. Based on these observations, we hypothesized that A2AR activation would ameliorate cartilage senescence. We found that A2AR stimulation of chondrocytes reduced beta-galactosidase staining and regulated levels and cell localization of common senescence mediators p21 and p16 in vitro in the human TC28a2 chondrocyte cell line. In vivo analysis similarly showed A2AR activation reduced nuclear p21 and p16 in obesity-induced OA mice injected with liposomal-CGS21680 and increased nuclear p21 and p16 in A2AR knockout mouse chondrocytes compared to wild-type mice. A2AR agonism also increased activity of the chondrocyte Sirt1/AMPK energy-sensing pathway by enhancing nuclear Sirt1 localization and upregulating T172-phosphorylated (active) AMPK protein levels. Lastly, A2AR activation in TC28a2 and primary human chondrocytes reduced wild-type p53 and concomitantly increased p53 alternative splicing leading to increase in an anti-senescent p53 variant, Δ133p53α. The results reported here indicate that A2AR signaling promotes chondrocyte homeostasis in vitro and reduces OA cartilage development in vivo by reducing chondrocyte senescence.

Background: Early surgical exposure and research fellowships can influence medical students' specialty choice, increase academic productivity, and impact residency match. However, to our knowledge, there is no published guidance on the programmatic evaluation and quality enhancement necessary for the sustainability of formal plastic surgery summer research programs for first year medical students. We present seven years (2013-2020) of institutional experience in an effort to inform program development at other institutions. Methods: From 2013 to 2016, a sole basic science research arm existed. In 2017, a clinical research arm was introduced, with several supplemental activities, including surgical skills curriculum. A formalized selection process was instituted in 2014. Participant feedback was analyzed annually. Long-term outcomes included continued research commitment, productivity, and residency match. Results: The applicant pool reached 96 applicants in 2019, with 85% from outside institutions. Acceptance rate reached 7% in 2020. With adherence to a scoring rubric for applicant evaluation, good to excellent interrater reliability was achieved (intraclass correlation coefficient = 0.75). Long-term outcomes showed that on average per year, 28% of participants continued involvement in departmental research and 29% returned for dedicated research. Upon finishing medical school, participants had a mean of 7 ± 4 peer-reviewed publications. In total, 62% of participants matched into a surgical residency program, with 54% in integrated plastic surgery. Conclusions: A research program designed for first year medical students interested in plastic surgery can achieve academic goals. Students are provided with mentorship, networking opportunities, and tools for self-guided learning and career development.

SIGNIFICANCE/CONCLUSIONS:Skin wounds and disorders compromise the protective functions of skin and patient quality of life. Though accessible on the surface, they are challenging to address due to paucity of effective therapies. Exogenous extracellular vesicles (EVs), cell-free derivatives of adult multipotent stromal cells (MSCs), are developing as a treatment modality. Knowledge of origin MSCs, EV processing and mode of action is necessary for directed use of EVs in preclinical studies and methodical translation. Recent advances: Nano to microscale EVs, though from non-skin cells, induce functional responses in cutaneous wound cellular milieu. EVs allow a shift from cell-based to cell-free/derived modalities by carrying the MSC beneficial factors but eliminating risks associated with MSC transplantation. EVs have demonstrated striking efficacy in resolution of preclinical wound models, specifically within the complexity of skin structure and wound pathology. CRITICAL ISSUES/RESULTS:To facilitate comparison across studies, tissue sources and processing of MSCs, culture conditions, isolation and preparations of EVs, and vesicle sizes require standardization as these criteria influence EV types and contents, and potentially determine the induced biological responses. Procedural parameters for all steps preceding the actual therapeutic administration may be the key to generating EVs that demonstrate consistent efficacy through known mechanisms. We provide a comprehensive review of such parameters and the subsequent tissue, cellular and molecular impact of the derived EVs in different skin wounds/disorders. FUTURE DIRECTIONS/CONCLUSIONS:We will gain more complete knowledge of EV-induced effects in skin, and specificity for different wounds/conditions. The safety and efficacy of current preclinical xenogenic applications will favor translation into allogenic clinical applications of EVs as a biologic.