Immune modulation in transplant medicine: a comprehensive review of cell therapy applications and future directions.

Balancing the immune response after solid organ transplantation (SOT) and vascularized composite allotransplantation (VCA) remains an ongoing clinical challenge. While immunosuppressants can effectively reduce acute rejection rates following transplant surgery, some patients still experience recurrent acute rejection episodes, which in turn may progress to chronic rejection. Furthermore, these immunosuppressive regimens are associated with an increased risk of malignancies and metabolic disorders. Despite significant advancements in the field, these IS related side effects persist as clinical hurdles, emphasizing the need for innovative therapeutic strategies to improve transplant survival and longevity. Cellular therapy, a novel therapeutic approach, has emerged as a potential pathway to promote immune tolerance while minimizing systemic side-effects of standard IS regiments. Various cell types, including chimeric antigen receptor T cells (CAR-T), mesenchymal stromal cells (MSCs), regulatory myeloid cells (RMCs) and regulatory T cells (Tregs), offer unique immunomodulatory properties that may help achieve improved outcomes in transplant patients. This review aims to elucidate the role of cellular therapies, particularly MSCs, T cells, Tregs, RMCs, macrophages, and dendritic cells in SOT and VCA. We explore the immunological features of each cell type, their capacity for immune regulation, and the prospective advantages and obstacles linked to their application in transplant patients. An in-depth outline of the current state of the technology may help SOT and VCA providers refine their perioperative treatment strategies while laying the foundation for further trials that investigate cellular therapeutics in transplantation surgery.

Bioengineering of vascularized porcine flaps using perfusion-recellularization.

Large volume soft tissue defects greatly impact patient quality of life and function while suitable repair options remain a challenge in reconstructive surgery. Engineered flaps could represent a clinically translatable option that may circumvent issues related to donor site morbidity and tissue availability. Herein, we describe the regeneration of vascularized porcine flaps, specifically of the omentum and tensor fascia lata (TFL) flaps, using a tissue engineering perfusion-decellularization and recellularization approach. Flaps were decellularized using a low concentration sodium dodecyl sulfate (SDS) detergent perfusion to generate an acellular scaffold with retained extracellular matrix (ECM) components while removing underlying cellular and nuclear contents. A perfusion-recellularization strategy allowed for seeding of acellular flaps with a co-culture of human umbilical vein endothelial cell (HUVEC) and mesenchymal stromal cells (MSC) onto the decellularized omentum and TFL flaps. Our recellularization technique demonstrated evidence of intravascular cell attachment, as well as markers of endothelial and mesenchymal phenotype. Altogether, our findings support the potential of using bioengineered porcine flaps as a novel, clinically-translatable strategy for future application in reconstructive surgery.

Human-Induced Pluripotent Stem Cells in Plastic and Reconstructive Surgery.

A hallmark of plastic and reconstructive surgery is restoring form and function. Historically, tissue procured from healthy portions of a patient's body has been used to fill defects, but this is limited by tissue availability. Human-induced pluripotent stem cells (hiPSCs) are stem cells derived from the de-differentiation of mature somatic cells. hiPSCs are of particular interest in plastic surgery as they have the capacity to be re-differentiated into more mature cells, and cultured to grow tissues. This review aims to evaluate the applications of hiPSCs in the plastic surgery context, with a focus on recent advances and limitations. The use of hiPSCs and non-human iPSCs has been researched in the context of skin, nerve, vasculature, skeletal muscle, cartilage, and bone regeneration. hiPSCs offer a future for regenerated autologous skin grafts, flaps comprised of various tissue types, and whole functional units such as the face and limbs. Also, they can be used to model diseases affecting tissues of interest in plastic surgery, such as skin cancers, epidermolysis bullosa, and scleroderma. Tumorigenicity, immunogenicity and pragmatism still pose significant limitations. Further research is required to identify appropriate somatic origin and induction techniques to harness the epigenetic memory of hiPSCs or identify methods to manipulate epigenetic memory.

Adipose-Derived Stem Cells: Angiogenetic Potential and Utility in Tissue Engineering.

Adipose tissue (AT) is a large and important energy storage organ as well as an endocrine organ with a critical role in many processes. Additionally, AT is an enormous and easily accessible source of multipotent cell types used in our day for all types of tissue regeneration. The ability of adipose-derived stem cells (ADSCs) to differentiate into other types of cells, such as endothelial cells (ECs), vascular smooth muscle cells, or cardiomyocytes, is used in tissue engineering in order to promote/stimulate the process of angiogenesis. Being a key for future successful clinical applications, functional vascular networks in engineered tissue are targeted by numerous in vivo and ex vivo studies. The article reviews the angiogenic potential of ADSCs and explores their capacity in the field of tissue engineering (TE).

Mesenchymal cells support the early retention of primary alveolar type 2 cells on acellular mouse lung scaffolds.

Objectives: Tissue engineering approaches via repopulation of acellular biological grafts provide an exciting opportunity to generate lung grafts for transplantation. Alveolar type 2 (AT2) cells are a promising cell source for re-epithelialization. There are however inherent limitations with respect to their survival and growth, thus impeding their usability for tissue engineering applications. This study investigates the use of mesenchymal stromal cells to support primary AT2 cells for recellularization of mouse lung scaffolds. Methods: AT2 cells and bone marrow-derived mesenchymal cells (BMC) were co-delivered to decellularized mouse lung scaffolds. Recellularized lungs were evaluated for cell surface coverage, viability, and differentiation at 1 and 4 days after cell seeding. Recellularization was evaluated via histological analysis and immunofluorescence. Results: Simultaneous delivery of AT2 and BMC into acellular lung scaffolds resulted in enhanced cell surface coverage and reduced AT2 cell apoptosis in the recellularized scaffolds at Day 1 but not Day 4. AT2 cell number decreased after 4 days in both of AT2 only and codelivery groups suggesting limited expansion potential in the scaffold. After retention in the scaffold, AT2 cells differentiated into Aqp5-expressing cells. Conclusions: Our results indicate that BMC support AT2 cell survival during the initial attachment and engraftment phase of recellularization. While our findings suggest only a short-term beneficial effect of BMC, our study demonstrates that AT2 cells can be delivered and retained in acellular lung scaffolds; thus with preconditioning and supporting cells, may be used for re-epithelialization. Selection and characterization of appropriate cell sources for use in recellularization, will be critical for ultimate clinical application.

Evaluation of a decellularized bronchial patch transplant in a porcine model.

Biological scaffolds for airway reconstruction are an important clinical need and have been extensively investigated experimentally and clinically, but without uniform success. In this study, we evaluated the use of a decellularized bronchus graft for airway reconstruction. Decellularized left bronchi were procured from decellularized porcine lungs and utilized as grafts for airway patch transplantation. A tracheal window was created and the decellularized bronchus was transplanted into the defect in a porcine model. Animals were euthanized at 7 days, 1 month, and 2 months post-operatively. Histological analysis, immunohistochemistry, scanning electron microscopy, and strength tests were conducted in order to evaluate epithelialization, inflammation, and physical strength of the graft. All pigs recovered from general anesthesia and survived without airway obstruction until the planned euthanasia timepoint. Histological and electron microscopy analyses revealed that the decellularized bronchus graft was well integrated with native tissue and covered by an epithelial layer at 1 month. Immunostaining of the decellularized bronchus graft was positive for CD31 and no difference was observed with immune markers (CD3, CD11b, myeloperoxidase) at two months. Although not significant, tensile strength was decreased after one month, but recovered by two months. Decellularized bronchial grafts show promising results for airway patch reconstruction in a porcine model. Revascularization and re-epithelialization were observed and the immunological reaction was comparable with the autografts. This approach is clinically relevant and could potentially be utilized for future applications for tracheal replacement.

Three Ds for diagnosing necrotizing fasciitis by front-line clinicians.

INTRODUCTION: Necrotizing fasciitis (NF) is a life-threatening infection and a surgical emergency. Not all clinicians have the experience or resources to detect NF in its early stages. OBJECTIVE: To develop a diagnostic algorithm for primary care and emergency physicians to identify patients with possible NF, including an initial approach to triaging such individuals. METHODS: Medline was searched to identify studies of validated algorithms for NF diagnosis and/or cohort or case series providing clinical and diagnostic features of NF. Candidate algorithms were validated via application to 3 published cases of initially misdiagnosed NF. We retrospectively reviewed NF cases between 2011 and 2022 at our center to validate our algorithm. RESULTS: The search yielded 540 articles; 109 were included following a review of abstracts. No published validated diagnostic algorithm was identified. Using the reported clinical and diagnostic features of NF, we generated an algorithm of the "3Ds" of NF: Disproportionate pain, Dermatological findings, and Disorganized physiology. A larger number of Ds indicated a greater level of suspicion for NF and prioritization for urgent surgical consultation. In 3 published cases of missed NF, the 3Ds algorithm successfully identified all as having possible NF. On reviewing our cases, we identified 56 patients with NF during an 11 year period. 66% of whom had the 3Ds at their initial presentation. DISCUSSION: The 3Ds algorithm, a simple and easy-to-remember tool can be easily applied in a primary or emergency care setting, and may improve the early diagnosis of NF. Retrospective analysis of NF cases allows for validation of this algorithm. However, this algorithm requires prospective validation.

Intraoperative vasopressor use does not increase complications in microvascular post-mastectomy breast reconstruction: Experience in 1729 DIEP flaps at a single center.

INTRODUCTION: Vasopressors are traditionally avoided in microsurgery due to concerns about their effect on free flap survival. We examine the impact of intraoperative vasopressors on microsurgical outcomes in a large series of DIEP flap breast reconstructions. METHODS: A retrospective chart review was performed of patients who underwent DIEP breast reconstruction between January 2010 and May 2020. Intraoperative and postoperative microsurgical outcomes were compared in patients who received vasopressors and those who did not. RESULTS: The study included 1102 women who underwent 1729 DIEP. 878 patients (79.7%) received intraoperative phenylephrine, ephedrine, or a combination of both. There was no significant difference in overall complications, intraoperative microvascular events, takebacks for microvascular complications, or partial or total flap loss between groups. Outcomes were not affected by vasopressor type, dose, or timing of administration. The vasopressor group received significantly lower intraoperative fluid volumes. Multivariate logistic regression found a significant association between overall complications and excessive fluids (OR 2.03, 99% CI 0.98-5.18, p = 0.03) but not vasopressor use (OR 0.79, 99% CI 0.64-3.16, p = 0.7) CONCLUSION: This study demonstrates that vasopressors do not adversely affect clinical outcomes after DIEP breast reconstruction. Withholding vasopressors results in excessive intravenous fluid administration and increased postoperative complications.

Ensuring patient centeredness in upper extremity lymphedema research: Identifying patient-prioritized agenda and preferences.

PURPOSE: To elicit a patient-prioritized agenda and preferences for upper extremity lymphedema (LE) research. METHODS: Focus group sessions (FGs) were conducted with English-speaking, adult women (18 years and older) with breast cancer-related LE (BCRL) seeking conservative or surgical care at two tertiary cancer centers in Ontario, Canada. An interview guide was used; women were asked to describe health-related quality of life (HRQL) outcomes that mattered the most to them, followed by their preferences for research study design and for providing patient-reported outcomes measure (PROM) data. Inductive content analysis was used to identify themes and subthemes. RESULTS: A total of 16 women participated in 4 FG sessions (55 ± 9.5 years) and described the impact of LE on their appearance, physical, psychosocial, and sexual well-being. Women emphasized that psychosocial well-being was often not discussed in clinical care and that they were poorly informed of LE risk and care options. Most women said that they would not be willing to be randomized to surgical versus conservative management of LE. They also expressed a preference to complete PROM data electronically. All women emphasized the value of having an open text option alongside PROMs to expand on their concerns. CONCLUSION: Patient centeredness is key to generating meaningful data and ensuring ongoing engagement in clinical research. In LE, comprehensive PROMs that measure a range of HRQL concerns, especially psychosocial well-being, should be considered. Women with BCRL are reluctant to be randomized to conservative care when a surgical option is available, resulting in implications for planning trial sample size and recruitment.

Immunohistochemical Analysis of Lymphocyte Populations in Acute Skin Rejection: The University Health Network Addition to the Banff Classification.

Acute rejection in vascularized composite allotransplantation has been identified using the Banff 2007 working classification. We propose an addition to this classification based on histological and immunological assessment within the skin and subcutaneous tissue. Methods: Biopsies from vascularized composite transplant patients were obtained at scheduled visits and whenever skin changes occurred. Histology and immunohistochemistry were performed on all samples, looking at infiltrating cells. Results: Observations were made specifically related to each component of the skin, including the epidermis, dermis, vessels, and subcutaneous tissue. Our findings led to the establishment of the University Health Network addition of skin rejection. Conclusions: The high rate of rejection where the skin is involved requires novel techniques for early detection. The University Health Network skin rejection addition can serve as an adjunct to the Banff classification.

Perfusion decellularization for vascularized composite allotransplantation.

Vascularized composite allotransplantation is becoming the emerging standard for reconstructive surgery treatment for patients with limb trauma and facial injuries involving soft tissue loss. Due to the complex immunogenicity of composite grafts, patients who undergo vascularized composite allotransplantation are reliant on lifelong immunosuppressive therapy. Decellularization of donor grafts to create an extracellular matrix bio-scaffold provides an immunomodulatory graft that preserves the structural and bioactive function of the extracellular matrix. Retention of extracellular matrix proteins, growth factors, and signaling cascades allow for cell adhesion, migration, proliferation, and tissue regeneration. Perfusion decellularization of detergents through the graft vasculature allows for increased regent access to all tissue layers, and removal of cellular debris through the venous system. Grafts can subsequently be repopulated with appropriate cells through the vasculature to facilitate tissue regeneration. The present work reviews methods of decellularization, process parameters, evaluation of adequate cellular and nuclear removal, successful applications of perfusion decellularization for use in vascularized composite allotransplantation, and current limitations.

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor.

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient's quality of life. Although surgical reconstruction can be performed using autologous free flap transfer or vascularized composite allotransplantation (VCA), such methods also have disadvantages. Issues such as donor site morbidity and tissue availability limit autologous free flap transfer, while immunosuppression is a significant limitation of VCA. Engineered tissues in reconstructive surgery using decellularization/recellularization methods represent a possible solution. Decellularized tissues are generated using methods that remove native cellular material while preserving the underlying extracellular matrix (ECM) microarchitecture. These acellular scaffolds can then be subsequently recellularized with recipient-specific cells. This protocol details the procurement and decellularization methods used to achieve acellular scaffolds in a pig model. In addition, it also provides a description of the perfusion bioreactor design and setup. The flaps include the porcine omentum, tensor fascia lata, and the radial forearm. Decellularization is performed via ex vivo perfusion of low concentration sodium dodecyl sulfate (SDS) detergent followed by DNase enzyme treatment and peracetic acid sterilization in a customized perfusion bioreactor. Successful tissue decellularization is characterized by a white-opaque appearance of flaps macroscopically. Acellular flaps show the absence of nuclei on histological staining and a significant reduction in DNA content. This protocol can be used efficiently to generate decellularized soft tissue scaffolds with preserved ECM and vascular microarchitecture. Such scaffolds can be used in subsequent recellularization studies and have the potential for clinical translation in reconstructive surgery.

Systematic Review and Guidelines for Perioperative Management of Pediatric Patients Undergoing Major Plastic Surgery Procedures, with a Focus on Free Tissue Transfer.

BACKGROUND: Microsurgical free tissue transfer has been successfully implemented for various reconstructive applications in children. The goal of this study was to identify the best available evidence on perioperative management of pediatric patients undergoing free tissue transfer and to use it to develop evidence-based care guidelines. METHODS: A systematic review was conducted in the PubMed, Embase, Scopus, and Cochrane Library databases. Because a preliminary search of the pediatric microsurgical literature yielded scant data with a low level of evidence, pediatric anesthesia guidelines for healthy children undergoing major operations were also included. Exclusion criteria included vague descriptions of perioperative care, case reports, and studies of syndromic or chronically ill children. RESULTS: Two hundred four articles were identified, and 53 met inclusion criteria. Management approaches specific to the pediatric population were used to formulate recommendations. High-quality data were found for anesthesia, analgesia, fluid administration/blood transfusion, and anticoagulation (Level I Evidence). Lower quality evidence was identified for patient temperature (Level III Evidence) and vasodilator use (Level IV Evidence). Key recommendations include administering sevoflurane for general anesthesia, implementing a multimodal analgesia strategy, limiting preoperative fasting, restricting blood transfusions until hemoglobin level is less than 7 g/dl unless the patient is symptomatic, and reserving chemical venous thromboembolism prophylaxis for high-risk patients. CONCLUSIONS: Pediatric-specific guidelines are important, as they acknowledge physiologic differences in children, which may be overlooked when extrapolating from adult studies. These evidence-based recommendations are a key first step toward standardization of perioperative care of pediatric patients undergoing plastic surgical procedures, including free tissue transfer, to improve outcomes and minimize complications.

Procurement and Decellularization of Rat Hindlimbs using an Ex Vivo Perfusion-based Bioreactor for Vascularized Composite Allotransplantation.

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an evolving reconstructive avenue for transferring multiple tissues as a composite subunit. Despite the promising nature of VCA, the long-term immunosuppressive requirements are a significant limitation due to the increased risk of malignancies, end-organ toxicity, and opportunistic infections. Tissue engineering of acellular composite scaffolds is a potential alternative in reducing the need for immunosuppression. Herein, the procurement of a rat hindlimb and its subsequent decellularization using sodium dodecyl sulfate (SDS) is described. The procurement strategy presented is based upon the common femoral artery. A machine perfusion-based bioreactor system was constructed and used for ex vivo decellularization of the hindlimb. Successful perfusion decellularization was performed, resulting in a white translucent-like appearance of the hindlimb. An intact, perfusable, vascular network throughout the hindlimb was observed. Histological analyses showed the removal of nuclear contents and the preservation of tissue architecture across all tissue compartments.

Recellularization of Bioengineered Scaffolds for Vascular Composite Allotransplantation.

Traumatic injuries or cancer resection resulting in large volumetric soft tissue loss requires surgical reconstruction. Vascular composite allotransplantation (VCA) is an emerging reconstructive option that transfers multiple, complex tissues as a whole subunit from donor to recipient. Although promising, VCA is limited due to side effects of immunosuppression. Tissue-engineered scaffolds obtained by decellularization and recellularization hold great promise. Decellularization is a process that removes cellular materials while preserving the extracellular matrix architecture. Subsequent recellularization of these acellular scaffolds with recipient-specific cells can help circumvent adverse immune-mediated host responses and allow transplantation of allografts by reducing and possibly eliminating the need for immunosuppression. Recellularization of acellular tissue scaffolds is a technique that was first investigated and reported in whole organs. More recently, work has been performed to apply this technique to VCA. Additional work is needed to address barriers associated with tissue recellularization such as: cell type selection, cell distribution, and functionalization of the vasculature and musculature. These factors ultimately contribute to achieving tissue integration and viability following allotransplantation. The present work will review the current state-of-the-art in soft tissue scaffolds with specific emphasis on recellularization techniques. We will discuss biological and engineering process considerations, technical and scientific challenges, and the potential clinical impact of this technology to advance the field of VCA and reconstructive surgery.

Evaluation of Early Markers of Ischemia-reperfusion Injury and Preservation Solutions in a Modified Hindlimb Model of Vascularized Composite Allotransplantation.

BACKGROUND: Ischemia-reperfusion injury plays an important role in vascularized composite allotransplantation (VCA). Currently, there is no ideal preservation solution for VCA. In this study, we investigated the effects of 4 different preservation solutions on different tissues within an allogeneic hindlimb rat model. METHODS: Sprague Dawley rat hindlimbs were flushed and placed at 4°C for 6 h in heparinized saline, histidine-tryptophan-ketoglutarate, University of Wisconsin (UW), and Perfadex and heterotopically transplanted for ease of ambulation. Apoptosis, necrosis, and the extracellular matrix of the tissues within the allograft were analyzed 2 h posttransplantation using immunohistochemistry, terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick-end labeling (TUNEL) assay, and enzyme-linked immunoassay. RESULTS: Higher expression of cleaved caspase 3, a significant increase of high-mobility group box 1 and TUNEL-positive apoptotic cells were observed in the muscle and vessels preserved with heparinized saline compared with UW and Perfadex following reperfusion. Higher expression of TUNEL-positive apoptotic cells was observed in the skin at 12 h of ischemia and in the nerve following reperfusion with histidine-tryptophan-ketoglutarate as a preservation solution. CONCLUSIONS: Our data suggest that UW and Perfadex are preferred solutions in VCA. The vessels within the allografts appear to be very susceptible, with laminins and CD31 playing a role in ischemia-reperfusion injury.

Bioreactor-Based De-epithelialization of Long-Segment Tracheal Grafts.

Tissue engineering techniques to generate a graft ex vivo is an exciting field of research. In particular, the use of biological scaffolds has shown to be promising in a clinical setting. In this approach, decellularized donor scaffolds are obtained following detergent-based enzymatic treatment to remove donor cells and subsequently repopulated with recipient specific cells. Herein, we describe our bioreactor-based partial decellularization approach to generate hybrid tracheal grafts. Using a short detergent-based treatment with sodium dodecyl sulfate (SDS), we remove the epithelium and maintain the structural integrity of the donor grafts by keeping the cartilage alive. The following will be a step-by-step description of the bioreactor system setup and partial decellularization protocol to obtain a de-epithelialized tracheal graft.

Short-Term Preclinical Application of Functional Human Induced Pluripotent Stem Cell-Derived Airway Epithelial Patches.

Airway pathologies including cancer, trauma, and stenosis lack effective treatments, meanwhile airway transplantation and available tissue engineering approaches fail due to epithelial dysfunction. Autologous progenitors do not meet the clinical need for regeneration due to their insufficient expansion and differentiation, for which human induced pluripotent stem cells (hiPSCs) are promising alternatives. Airway epithelial patches are engineered by differentiating hiPSC-derived airway progenitors into physiological proportions of ciliated (73.9 ± 5.5%) and goblet (2.1 ± 1.4%) cells on a silk fibroin-collagen vitrigel membrane (SF-CVM) composite biomaterial for transplantation in porcine tracheal defects ex vivo and in vivo. Evaluation of ex vivo tracheal repair using hiPSC-derived SF-CVM patches demonstrate native-like tracheal epithelial metabolism and maintenance of mucociliary epithelium to day 3. In vivo studies demonstrate SF-CVM integration and maintenance of airway patency, showing 80.8 ± 3.6% graft coverage with an hiPSC-derived pseudostratified epithelium and 70.7 ± 2.3% coverage with viable cells, 3 days postoperatively. The utility of bioengineered, hiPSC-derived epithelial patches for airway repair is demonstrated in a short-term preclinical survival model, providing a significant leap for airway reconstruction approaches.

Preservation solutions for attenuation of ischemia-reperfusion injury in vascularized composite allotransplantation.

Vascularized composite allotransplantation represents the final level of the reconstructive ladder, offering treatment options for severe tissue loss and functional deficiencies. Vascularized composite allotransplantation is particularly susceptible to ischemia-reperfusion injury and requires preservation techniques when subjected to extended storage times prior to transplantation. While static cold storage functions to reduce ischemic damage and is widely employed in clinical settings, there exists no consensus on the ideal preservation solution for vascularized composite allotransplantation. This review aims to highlight current clinical and experimental advances in preservation solution development and their critical role in attenuating ischemia-reperfusion injury in the context of vascularized composite allotransplantation.