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Introduction: Multiple perfusion systems have been investigated on vascularized composite allografts, with various temperatures and different preservation solutions, most using continuous flow (CF). However, physiological flow is pulsatile and provides better outcomes in kidney and lung ex vivo perfusions. The objective of this pilot study is to compare pulsatile flow (PF) with CF in our 24-h subnormothermic machine perfusion protocol for swine hindlimbs., Methods: Partial hindlimbs were harvested from Yorkshire pigs and perfused with a modified Steen solution at 21°C for 24 h either with CF (n = 3) or with pulsatile flow (PF) at 60 beats/min (n = 3). Perfusion parameters, endothelial markers, and muscle biopsies were assessed at different timepoints., Results: Overall, lactate levels were significantly lower in the PF group (P = 0.001). Glucose uptake and potassium concentration were similar in both groups throughout perfusion. Total nitric oxide levels were significantly higher in the PF group throughout perfusion (P = 0.032). Nitric oxide/endothelin-1 ratio also tends to be higher in the PF group, reflecting a potentially better vasoconductivity with PF, although not reaching statistical significance (P = 0.095). Arterial resistances were higher in the PF group (P < 0.001). Histological assessment did not show significant difference in muscular injury between the two groups. Weight increased quicker in the CF group but reached similar values with the PF after 24 h., Conclusions: This pilot study suggests that PF may provide superior preservation of vascularized composite allografts when perfused for 24 h at subnormothermic temperatures, with potential improvement in endothelial function and decreased ischemic injury., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Machine perfusion is gaining interest as an efficient method of tissue preservation of Vascularized Composite Allografts (VCA). The aim of this study was to develop a protocol for ex vivo subnormothermic oxygenated machine perfusion (SNMP) on rodent hindlimbs and to validate our protocol in a heterotopic hindlimb transplant model., Methods: In this optimization study we compared three different solutions during 6 h of SNMP (n = 4 per group). Ten control limbs were stored in a preservation solution on Static Cold Storage [SCS]). During SNMP we monitored arterial flowrate, lactate levels, and edema. After SNMP, muscle biopsies were taken for histology examination, and energy charge analysis. We validated the best perfusion protocol in a heterotopic limb transplantation model with 30-d follow up (n = 13). As controls, we transplanted untreated limbs (n = 5) and hindlimbs preserved with either 6 or 24 h of SCS (n = 4 and n = 5)., Results: During SNMP, arterial outflow increased, and lactate clearance decreased in all groups. Total edema was significantly lower in the HBOC-201 group compared to the BSA group (P = 0.005), 4.9 (4.3-6.1) versus 48.8 (39.1-53.2) percentage, but not to the BSA + PEG group (P = 0.19). Energy charge levels of SCS controls decreased 4-fold compared to limbs perfused with acellular oxygen carrier HBOC-201, 0.10 (0.07-0.17) versus 0.46 (0.42-0.49) respectively (P = 0.002)., Conclusions: Six hours ex vivo SNMP of rodent hindlimbs using an acellular oxygen carrier HBOC-201 results in superior tissue preservation compared to conventional SCS., (Copyright © 2021. Published by Elsevier Inc.)

Background: The autologous vein remains the standard conduit for lower extremity and coronary artery bypass grafting despite a 30%-50% 5-y failure rate, primarily attributable to intimal hyperplasia (IH) that develops in the midterm period (3-24 mo) of graft maturation. Our group discovered that externally strengthening vein grafts by cross-linking the adventitial collagen with photochemical tissue passivation (PTP) mitigates IH in an arteriovenous model at 4 wk. We now investigate whether this effect is retained in the midterm period follow-up., Methods: Six Hanford miniature pigs received bilateral carotid artery interposition vein grafts. In each animal, the external surface of one graft was treated with PTP before grafting, whereas the opposite side served as the untreated control. The grafts were harvested after 3 mo. Ultrasound evaluation of all vein grafts was performed at the time of grafting and harvest. The grafts were also evaluated histomorphometrically and immunohistologically for markers of IH., Results: All vein grafts were patent at 3 mo except one graft in the PTP-treated group because of early technical failure. The control vein grafts had significantly greater IH than PTP-treated grafts at 3 mo, as evidenced by the intimal area (2.6 ± 1.0 mm 2 versus 1.4 ± 1.5 mm 2 , respectively, P = 0.045) and medial area (5.1 ± 1.9 mm 2 versus 2.7 ± 2.4 mm 2 , respectively, P = 0.048). The control grafts had an increased presence and proliferation of mural myofibroblasts with greater smooth muscle actin and proliferating cell nuclear antigen staining., Conclusions: PTP treatment to the external surface of the vein grafts decreases IH at 3 mo after arteriovenous grafting and may prevent future graft failure., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Limited options exist for the restoration of craniofacial cartilage. Autologous tissue or porous polyethylene is currently used for nasal and auricular reconstruction. Both options are associated with drawbacks, including donor site morbidity and implant extrusion. Poly(vinyl alcohol) (PVA) is a non-degradable flexible biocompatible polymer than can be engineered to mimic the properties of cartilage. The goal of this study was to engineer a biosynthetic hybrid construct using a combination of PVA-alginate hydrogels and human nasal septum chondrocytes., Materials and Methods: Chondrocytes isolated from human nasal septum cartilage were expanded and mixed with 2% sodium alginate hydrogel. The chondrocyte-alginate mix was injected into a non-degradable porous PVA hydrogel, creating biosynthetic constructs. A group of these constructs were implanted into the subcutaneous environment of nude mice, while the other group was cultured in a spinner flask bioreactor system for 10 d and then implanted. After 6 wk in vivo, the histologic, biochemical, and biomechanical properties were examined., Results: Histological analysis demonstrated sulfated glycosaminoglycans and deposition of collagen type II in constructs from both groups. Constructs cultured in the bioreactor system prior in vivo implantation demonstrated higher levels of DNA, glycosaminoglycans, and hydroxyproline. An increase of 22% in the compressive strength of the engineered constructs exposed to the bioreactor was also observed., Conclusion: A novel porous PVA-alginate gel hybrid was used to successfully engineer human cartilage in vivo. A 10-d period of bioreactor culturing increased levels of DNA, glycosaminoglycans, hydroxyproline, and the compressive modulus of the constructs., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: Ischemia reperfusion injury is a well-known phenomenon affecting skin flap viability. One method to improve flap viability is ischemic preconditioning. Previous murine flap models used random flaps. We developed a single pedicle island skin flap which allows us to create true ischemia by clamping the single pedicle. Our first aim was to describe a novel murine skin flap model with a definable, reproducible injury. Our second aim was to test the usefulness of this model by demonstrating mitigation of injury via ischemic preconditioning., Materials and Methods: Dorsal lateral thoracic artery pedicle island skin flaps (3.5 x 1.5 cm) were elevated in 39 male C57/BL6 mice: a Control group (n = 7), 10 h of ischemia (n = 21), and Preconditioning (2 cycles of 20 min ischemia: 20 minutes reperfusion) + 10-h ischemia (n = 11). After flap elevation, a silicon sheet barrier was placed. The axial pedicles were occluded, and the flaps were inset with 6-0 prolene. In all mice, ischemia was followed by 1 wk of reperfusion. At 1 wk, percent necrosis was measured and an analysis of variance was performed., Results: The percent of flap necrosis was 1.1% +/- 1.11% in controls. Animals that were subjected to 10 h of ischemia developed 33.14% +/- 7.23% necrosis. Preconditioned animals that underwent 10 h of ischemia demonstrated a 43% reduction in necrosis (18.82% +/- 5.68%). There was a statistically significant difference among all groups (P < or = 0.001)., Conclusion: Rat models have been the standard for skin flap experiments. We have developed a novel murine single pedicle island skin flap model with reproducible injury. This model has numerous advantages, including ease of handling, low cost, appropriateness for biomedical studies, and the availability of genetically altered animals. We also confirmed this model's usefulness in a study of mitigation of ischemia reperfusion injury through ischemic preconditioning.

Introduction: Peripheral nerve transection initiates a complex molecular response in the severed nerve endings, resulting in the release of neurotrophic and neurotropic factors that are central to axonal survival and regeneration. In this study we tested the hypothesis that sealing the neurorrhaphy site from the surrounding environment using a photochemically bonded nerve wrap would optimize the endoneural environment and enhance regeneration and nerve function recovery., Materials and Methods: Adult rats underwent unilateral sciatic nerve transection and standard epineural nerve repair. The repair site was wrapped with amniotic membrane or autologous vein and then was either sealed using photochemical tissue bonding (PTB) or secured with sutures. Photochemical sealing without a wrap was also carried out. Functional recovery was assessed at 2-wk intervals using walking track analysis and nerve histomorphometry was assessed at 12 wk., Results: Treating nerves with PTB-sealed amnion significantly improved functional recovery and increased distal axon and fiber diameters and myelin thickness compared to nerves treated with standard neurorrhaphy alone. Direct PTB sealing of the repair site also improved function. Neither amnion secured with sutures nor vein wraps exhibited improved functional or histological recovery compared to standard neurorrhaphy., Conclusions: These results suggest that sealing the peripheral nerve repair site with amnion using a photochemical technique may lead to earlier restoration of neural homeostasis and consequent enhanced repair of nerve injury.

Background: Ischemic preconditioning (IPC) mitigates ischemia-reperfusion (I/R) injury in experimental models. However, the clinical significance of this protection has been unclear and a mortality reduction has not been previously reported in noncardiac models. This study examined the local and remote protection afforded by skeletal muscle IPC and sought to determine the significance of this protection on mortality., Methods: Mice subjected to 2 h hindlimb ischemia/24 h reperfusion (standard I/R injury) were compared with those undergoing a regimen of two 20-min cycles of IPC followed by standard I/R injury. Local injury was assessed via gastrocnemius histology, and remote injury was evaluated via intestinal histology and pulmonary neutrophil infiltration (n = 7). Mortality was compared in parallel groups for 1 week (n = 6). Groups were analyzed using an unpaired Student's t-test for gastrocnemius and pulmonary injury, and a Mann-Whitney rank sum test for intestinal injury. Mortality differences were interpreted through a hazard ratio., Results: Significant protection was observed in preconditioned animals. There was a 35% local injury reduction in skeletal muscle (71.2% versus 46.0%, P < 0.01), a 50% reduction in remote intestinal injury (2.3 versus 1.1, P < 0.01), and a 43% reduction in remote pulmonary injury (14.9 versus 8.5, P < 0.01) compared with standard injury controls. Preconditioned animals were also significantly protected from mortality, demonstrating a 66.7% survival at 1 wk compared with 0% survival after standard injury alone (hazard ratio 0.20, 95% CI: 0.02-0.59)., Conclusions: We have developed a murine model of IPC that demonstrates local and remote protection against I/R injury, and exhibits significant mortality reduction. This model demonstrates the powerful effect of IPC on local and remote tissues and will facilitate further study of potential mechanisms and therapies.

Background: Photochemical tissue bonding (PTB) is a novel tissue repair technique that uses visible light and a photosensitizing dye to crosslink proteins on tissue surfaces. This technique has been successfully demonstrated in a number of tissue repair models. An ideal nerve repair technique would be atraumatic and avoid placement of foreign bodies at the repair site. The epineurium is suited to photochemical repair as it is thin, translucent and has a relatively high collagen content. This study was designed to determine if PTB could be successfully applied in a peripheral nerve repair model., Material and Methods: Forty Sprague Dawley rats underwent transection of the sciatic nerve. Animals were then randomized to four treatment groups; epineurial suture repair, epineurial cuff with PTB, epineurial cuff alone, and no repair. Functional recovery was assessed at 10 day intervals using walking track analysis and sciatic function index calculations. At 90 days postoperatively animals were sacrificed and sciatic nerves harvested for histology and histomorphometry., Results: Functional recovery in the suture repair and epineural cuff with PTB groups were not significantly different (-70.6 +/- 17.8 versus -76.9 +/- 10.3, P = 0.64) at 90 days postrepair. Histology showed good axonal regeneration with all repair techniques. Histomorphometric analysis found no significant difference between the repair groups., Conclusions: This study illustrates that peripheral nerves can be successfully repaired using a photochemical tissue bonding technique with results similar to those achieved with the current gold standard. With further development and refinement PTB may prove a useful tool in peripheral nerve repair.

Background: Isolated perfusion models can yield important data regarding metabolism of the skin. An effective model must remain stable during perfusion but respond appropriately to metabolic and vascular stimuli. We describe the design and characterization of a tubed superficial epigastric isolated perfusion flap., Materials and Methods: Tubed superficial epigastric flaps were created in 20 male Sprague Dawley rats. Forty-eight hours later the femoral vessels were cannulated and the flaps were perfused using a Krebs-Heinseleit buffer containing albumin for a period of 2 h. In five of the flaps norepinephrine and acetylcholine were added sequentially to the perfusate to determine vascular reactivity. In a further four flaps insulin (20 U/liter) and iodoacetate (5 mM) were added to the perfusate to confirm that the flap was metabolically active and reactive. Venous outflow was collected at regular intervals and analyzed for electrolytes, lactate, and glucose content. Vascularity and skin perfusion were characterized using barium microangiography and methylene blue dye injection., Results: This flap model was found to be stable in terms of arterial pressure, electrolyte levels, and lactate production over the perfusion period. Norepinephrine caused a sharp increase in vascular resistance, which was reversed by administration of acetylcholine. Lactate production increased appropriately with the addition of insulin to the perfusate with a rapid decline following addition of the glycolysis inhibitor iodoacetate. There was no leakage of perfusate or significant swelling of the flap during the perfusion., Conclusions: The tubed superficial epigastric artery flap makes an effective model for isolated perfusion studies of the skin with a wide range of experimental applications.

The ability to safely manipulate the immune system of the developing fetus carries the hope of effective treatment strategies for certain congenital disorders that can be diagnosed during gestation. One possible intervention is the induction of specific transplantation tolerance to an adult donor who could provide tissue after birth without the need for immunosuppression. Although the introduction of allogeneic stem cells to a developing immune system has been shown to result in hematopoietic chimerism, donor-specific transplantation tolerance has not been demonstrated in a large animal model. In previous reports of in utero stem-cell transplantation, the cells were injected into the fetus by an intraperitoneal route. We sought to improve upon this technique of cell transplantation by developing a method for the safe delivery of allogeneic stem cells directly into the hepatic circulation of fetal swine. In the second phase of our study, we determined if adult allogeneic bone marrow cells delivered to the fetus by this intravascular route could result in result in hematopoietic chimerism and donor-specific transplantation tolerance. A method of successful intravascular injection was designed in which a laparotomy was performed on a sow at midgestation (50-55 days) to administer 1 cc of inoculum into the portal vein of each fetus using transuterine ultrasound guidance and a 25-gauge spinal needle. In one sow, 10 piglets were injected with saline to test safety, and 8 piglets were born. For transplantation of stem cells to the fetuses, donor bone marrow was harvested from a genetically defined miniature swine. In one sow the marrow was injected without T-cell depletion resulting in abortion. In the third sow, the marrow was depleted of T-cells to less than 0.01% using magnetic beads conjugated to anti-CD3 monoclonal antibodies. No chimerism was detected in these offspring. Only in the fourth sow where the T-cell depletion was reduced to about 1% of the cells in the inoculum did one animal demonstrate chimerism. This piglet showed reproducible blood chimerism (0.95% donor cells) detected by flow cytometry measurement of monoclonal antibodies to the donor MHC. In addition, this animal demonstrated hyporesponsiveness to donor lymphocytes in an MLR assay while reacting strongly to third-party stimulator cells. A split-thickness skin graft from the donor was accepted, and a third-party graft was rapidly rejected., (Copyright 2000 Academic Press.)

Background: Success has been reported using intrathymic injection in the preconditioning regimen to induce allograft tolerance. Although long-term stable tolerance has been achieved in numerous rodent vascularized solid organ allograft models, tolerance to skin transplants has only been achieved across minor antigenic or concordant species disparities. This study sought to induce tolerance across an allogeneic barrier in a rat model with a major genetic disparity., Materials and Methods: Lewis rats were injected intrathymically with 1 x 10(8) Brown-Norway (BN) bone marrow cells and intraperitoneally with 1.0 cc of rabbit anti-rat anti-lymphocyte serum (ALS). Twenty-one days later, BN skin grafts were placed on the injected animals. Control groups were included to isolate the effect of technique, thymic manipulation, strain specificity, and ALS., Results: Animals receiving both intrathymic bone marrow cells and ALS had a skin graft median survival time of 24 days versus 8 days for the control group (P = 0.003). Groups receiving anti-lymphocyte serum alone or intrathymic bone marrow cell injection alone exhibited no skin graft survival prolongation. Mixed lymphocyte reactions revealed normal responsiveness of tolerant animal lymphocytes to donor strain lymphocytes., Conclusion: This protocol utilizing the intrathymic injection of donor bone marrow cells along with short-term immunosuppression with anti-lymphocyte serum produced markedly prolonged survival of skin allografts transplanted across a major histocompatibility barrier. Although tolerance was incomplete, significant prolongation has not previously been reported in genetic disparities of this degree. These results suggest that the application of this technique for central immune modulation may be beneficial for allograft tolerance induction and deserves further study in large animals models., (Copyright 1999 Academic Press.)

The clinical feasibility of transplantation of a vascularized limb tissue allograft depends upon reducing immunosuppression and its associated toxicity for the graft recipient. Donor or allograft irradiation would eradicate allogeneic marrow and provide a possible alternative or beneficial addition to host immunosuppression. The effect of irradiation on survival of limb tissue allograft was investigated in this study. In a rat model, knee allografts consisting of bone, cartilage, and soft tissues were transplanted across a strong histocompatibility barrier by femoral vascular microanastomoses. The grafts were harvested 1 and 2 weeks postoperatively for histologic and immunologic assays. Gamma irradiation of the graft immediately before transplant decreased graft rejection slightly. However, total body irradiation of the donor followed by a "waiting period" of 2 or more days before transplant significantly delayed rejection. After such a 6-day and a 2-day pretreatment protocol, the host cellular immune responses were not observed 1 and 2 weeks after transplantation and eventual progression toward graft rejection coincided with emergence of host antibody production. Rejection of vascularized limb tissue allografts after donor irradiation thus appeared to be mediated primarily through the humoral pathway. We conclude that donor irradiation is a potentially useful adjunct in prolonging survival of vascularized limb tissue allografts.