Faculty Bibliography

Vascularized composite allotransplantation (VCA) is increasingly recognized as a reconstructive option that offers substantial benefits for patients with severe tissue loss, including injuries affecting the face and hand. Despite its clinical promise, VCA remains limited by surgical complexity, the need for long-term immunosuppression, and associated morbidity. Preclinical models are therefore essential for refining surgical techniques, testing novel immunomodulatory approaches, and investigating ischemia/reperfusion injury. Compared with large-animal models, rodent models offer advantages in accessibility and cost while adhering more closely to the 3Rs principles of Replacement, Reduction, and Refinement. Among them, the mouse offers unique advantages, including the availability of humanized strains, a broad panel of well-characterized antibodies, and compatibility with advanced immunological assays. These features make the mouse particularly valuable for translational VCA research. Murine VCA, however, requires demanding supermicrosurgical skills, as vascular anastomoses are performed on vessels measuring approximately 0.3-0.5 mm in diameter. To enhance reproducibility, we provide technical tips that have proven critical to the success of these procedures. These include the insertion of a nylon filament into the vessel lumen to stabilize the anastomosis, careful separation of the artery and vein over a sufficient distance along the pedicle, precise anesthetic dosing, and specific maneuvers to prevent pedicle twisting. We also highlight common pitfalls and errors, offering practical guidance to improve outcomes. Here, we describe a comprehensive, stepwise protocol for mouse heterotopic hindlimb-to-neck transplantation, accompanied by instructional video material. This non-functional model minimizes postoperative morbidity compared with orthotopic transplantation while providing robust and reproducible results. It is ideally suited for training in supermicrosurgery and for addressing key experimental questions in VCA, including immune tolerance, graft preservation, and ischemia/reperfusion mechanisms. This methodology provides investigators with a reliable murine platform to advance translational VCA research and to develop innovative strategies aimed at improving outcomes in reconstructive transplantation.

Chronic wounds, especially in diabetic patients, pose a significant clinical challenge due to impaired microvasculature and delayed healing. This study presents Exo-Q, a novel thermoresponsive hydrogel formed by co-gelation of engineered Q protein nanofibers with exosomes, a class of vesicular intercellular communication mediators. Exo-Q transitions from a gel to a viscoelastic solution at physiological temperature, enabling localized, topical delivery of exosomes with an initial burst release followed by sustained release. In a diabetic mouse wound model, Exo-Q effectively delivered human bone marrow multipotent stromal cell-derived exosomes directly to the wound bed, where they accumulated in endothelial cells of granulation tissue without detectable systemic distribution. Exosomes produced under stringent and replicable cell culture conditions consistently carried biomacromolecular cargo enriched for miRNAs with validated targets in angiogenesis-associated genes, indicative of their therapeutic potential. Topical application of Exo-Q resulted in extensive neovascularized granulation tissue, significantly accelerating wound closure to levels comparable to non-diabetic wounds. Importantly, the hydrogel's modular design maintained the functional integrity of Q protein nanofibers and exosomes, demonstrating compatibility with full-thickness human wounds. This platform allows for tailored customization to address critical stages of diabetic wound healing while ensuring efficacy at low dosages, potentially enabling patient-administered treatment. By leveraging advanced biomaterials, Exo-Q advances the therapeutic efficacy of exosome-based interventions for diabetic wounds, offering a localized, non-invasive solution to chronic, non-healing wounds. This innovative hydrogel platform represents a modular therapeutic strategy with significant potential for clinical applications in regenerative medicine.

Vascularized composite allotransplantation (VCA) involves immunologically heterogeneous tissues with a high incidence of acute rejection. Reliable and timely detection of rejection onset remains a major unmet challenge in VCA management. This longitudinal exploratory case study assessed blood- and tissue-derived biomarkers for acute rejection monitoring in a full-face and bilateral hand transplant recipient over 4.6 years. Of these biomarkers, donor-derived cell-free DNA (dd-cfDNA) and short tandem repeats (STR) showed trends toward elevated recipient levels during acute rejection, though differences were not statistically significant. CD8⁺ T-cell percentages increased before acute rejection onset, highlighting a temporal association. Anti-angiotensin II type 1 receptor antibody (AT1R-Ab) levels did not differ significantly between acute rejection and non-rejection episodes, possibly due to prophylactic immune cell depletion. While dd-cfDNA and STR levels correlate with rejection episodes and reflect key graft cellular events, CD8⁺ T-cell dynamics demonstrated the strongest temporal association with rejection episodes in this patient, though no biomarker showed statistically significant differences. These exploratory findings support the need for further longitudinal, multi-patient studies to validate emerging biomarkers and refine rejection monitoring strategies in VCA.

Chronic wounds, especially in diabetic patients, pose a significant clinical challenge due to impaired microvasculature and delayed healing. This study presents Exo-Q, a novel thermoresponsive hydrogel formed by co-gelation of engineered Q protein nanofibers with exosomes, a class of vesicular intercellular communication mediators. Exo-Q transitions from a gel to a viscoelastic solution at physiological temperature, enabling localized, topical delivery of exosomes with an initial burst release followed by sustained release. In a diabetic mouse wound model, Exo-Q effectively delivered human bone marrow multipotent stromal cell-derived exosomes directly to the wound bed, where they accumulated in endothelial cells of granulation tissue without detectable systemic distribution. Exosomes produced under stringent and replicable cell culture conditions consistently carried biomacromolecular cargo enriched for miRNAs with validated targets in angiogenesis-associated genes, indicative of their therapeutic potential. Topical application of Exo-Q resulted in extensive neovascularized granulation tissue, significantly accelerating wound closure to levels comparable to non-diabetic wounds. Importantly, the hydrogel's modular design maintained the functional integrity of Q protein nanofibers and exosomes, demonstrating compatibility with full-thickness human wounds. This platform allows for tailored customization to address critical stages of diabetic wound healing while ensuring efficacy at low dosages, potentially enabling patient-administered treatment. By leveraging advanced biomaterials, Exo-Q advances the therapeutic efficacy of exosome-based interventions for diabetic wounds, offering a localized, non-invasive solution to chronic, non-healing wounds. This innovative hydrogel platform represents a modular therapeutic strategy with significant potential for clinical applications in regenerative medicine.

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.

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.

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.

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.