Orthotopic forelimb transplantation in a Yucatan minipig model: Anatomic and in vivo study.

INTRODUCTION: Above elbow transplants represent 19% of the upper extremity transplants. Previous large-animal models have been too distal or heterotopic, did not use immunosuppression and had short survival. We hypothesize that an orthotopic forelimb transplant model, under standard immunosuppression, is feasible and can be used to address questions on peri-transplant ischemia reperfusion injury, and post-transplantation vascular, immunologic, infectious, and functional outcomes. MATERIALS AND METHODS: Four forelimbs were used for anatomical studies. Four mock transplants were performed to establish technique/level of muscle/tendon repairs. Four donor and four recipient female Yucatan minipigs were utilized for in-vivo transplants (endpoint 90-days). Forelimbs were amputated at the midarm and preserved through ex vivo normothermic perfusion (EVNP) utilizing an RBC-based perfusate. Hourly perfusate fluid-dynamics, gases, electrolytes were recorded. Contractility during EVNLP was graded hourly using the Medical Research Council scale. EVNP termination criteria included systolic arterial pressure ≥115 mmHg, compartment pressure ≥30 mmHg (at EVNP endpoint), oxygen saturation reduction of 20%, and weight change ≥2%. Indocyanine green (ICG) angiography was performed after revascularization. Limb rejection was evaluated clinically (rash, edema, temperature), and histologically (BANFF classification) collecting per cause and protocol biopsies (POD 1, 7, 30, 60 and endpoint). Systemic infections were assessed by blood culture and tissue histology. CT scan was used to confirm bone bridging at endpoint. RESULTS: Animals 2, 4 reached endpoint with grade 0-I rejection. Limbs 1, 3 presented grade III rejection on days 6, 61. CsA troughs averaged 461 ± 189 ng/mL. EVNLP averaged 4.3 ± 0.52 h. Perfusate lactate, PO2 , and pH were 5.6 ± 0.9 mmol/L, 557 ± 72 mmHg and 7.5 ± 0.1, respectively. Muscle contractions were 4 [1] during EVNLP. Transplants 2, 3, 4 showed bone bridging on CT. CONCLUSION: We present preliminary evidence supporting the feasibility of an orthotopic, mid-humeral forelimb allotransplantation model under standard immunosuppression regimen. Further research should validate the immunological, infectious, and functional outcomes of this model.

Development of a reproducible porcine model of infected burn wounds.

Severe burns are traumatic and physically debilitating injuries with a high rate of mortality. Bacterial infections often complicate burn injuries, which presents unique challenges for wound management and improved patient outcomes. Currently, pigs are used as the gold standard of pre-clinical models to study infected skin wounds due to the similarity between porcine and human skin in terms of structure and immunological response. However, utilizing this large animal model for wound infection studies can be technically challenging and create issues with data reproducibility. We present a detailed protocol for a porcine model of infected burn wounds based on our experience in creating and evaluating full thickness burn wounds infected with Staphylococcus aureus on six pigs. Wound healing kinetics and bacterial clearance were measured over a period of 27 d in this model. Enumerated are steps to achieve standardized wound creation, bacterial inoculation, and dressing techniques. Systematic evaluation of wound healing and bacterial colonization of the wound bed is also described. Finally, advice on animal housing considerations, efficient bacterial plating procedures, and overcoming common technical challenges is provided. This protocol aims to provide investigators with a step-by-step guide to execute a technically challenging porcine wound infection model in a reproducible manner. Accordingly, this would allow for the design and evaluation of more effective burn infection therapies leading to better strategies for patient care.

Ex Vivo Normothermic Perfusion of Human Upper Limbs.

BACKGROUND: Ischemia-reperfusion injury remains a primary concern in upper extremity transplantation. Ex vivo normothermic perfusion (EVNP) enables near-physiological organ preservation, avoiding the deleterious effects of hypoxia and cooling. We investigated the effectiveness of human limb EVNP compared with static cold storage (SCS). METHODS: Twenty human upper extremities were procured. Ten were perfused at 38 °C with an oxygenated red blood cell-based solution, and contralateral limbs served as SCS control (4 °C). EVNP was terminated with systolic arterial pressure ≥115 mm Hg, compartment fullness, or a 20% decline in oxygen saturation. Weight, contractility, compartment pressure, tissue oxygen saturation, and uptake rates were assessed. Perfusate fluid dynamics, gases, electrolytes, and metabolites were measured. Myocyte injury scores and liquid chromatography-mass spectrometry analysis were performed. RESULTS: EVNP duration was 41.6 ± 9.4 h. Vascular resistance averaged 173.0 ± 29.4 mm Hg × min/L. Weight change and compartment pressures were 0.4 ± 12.2% ( P = 0.21) and 21.7 ± 15.58 mm Hg ( P = 0.003), respectively. Arterial and venous carbon dioxide partial pressure, oxygen saturation, and pH were 509.5 ± 91.4 mm Hg, 15.7 ± 30.2 mm Hg, 87.4 ± 11.4%, and 7.3 ± 0.2, respectively. Oxygen uptake rates averaged 5.7 ± 2.8 mL/min/g. Lactate reached 20 mmol/L after 15 (interquartile range = 6) h. Limb contractility was preserved for 30.5 (interquartile range = 15.8) h ( P < 0.001) and negatively correlated with perfusate potassium (ρ = -0.7, P < 0.001). Endpoint myocyte injury scores were 28.9 ± 11.5% (EVNP) and 90.2 ± 11.8% (SCS) ( P < 0.001). A significant increase in taurine ( P = 0.002) and decrease in tryptophan ( P = 0.002) were detected. Infrared thermography and indocyanine green angiography confirmed the presence of peripheral perfusion. CONCLUSIONS: EVNP can overcome the limitations of cold preservation by extending preservation times, enabling limb quality assessment, and allowing limb reconditioning before transplantation.

Metabolic Profiling of Skeletal Muscle During Ex-Vivo Normothermic Limb Perfusion.

INTRODUCTION: Ex vivo normothermic limb perfusion (EVNLP) provides several advantages for the preservation of limbs following amputation: the ability to maintain oxygenation and temperature of the limb close to physiological values, a perfusion solution providing all necessary nutrients at optimal concentrations, and the ability to maintain physiological pH and electrolytes. However, EVNLP cannot preserve the organ viability infinitely. We identified evidence of mitochondrial injury (swelling, elongation, and membrane disruption) after 24 hours of EVNLP of human upper extremities. The goal of this study was to identify metabolic derangements in the skeletal muscle during EVNLP. MATERIALS AND METHODS: Fourteen human upper extremities were procured from organ donors after family consent. Seven limbs underwent EVNLP for an average of 41.6 ± 9.4 hours, and seven contralateral limbs were preserved at 4°C for the same amount of time. Muscle biopsies were performed at 24 hours of perfusion, both from the EVNLP and control limbs. Perturbations in the metabolic profiles of the muscle during EVNLP were determined via untargeted liquid chromatography-mass spectrometry (MS) operated in positive and negative electrospray ionization modes, over a mass range of 50 to 750 Da. The data were deconvoluted using the XCMS software and further statistically analyzed using the in-house statistical package, MetaboLyzer. Putative identification of metabolites using exact mass within ±7 ppm mass error and MS/MS spectral matching to the mzCloud spectral library were performed via Compound Discoverer v.2.1 (Thermo Scientific, Fremont, CA, USA). We further validated the identity of candidate metabolites by matching the fragmentation pattern of these metabolites to those of their reference pure chemicals. A nonparametric Mann-Whitney U-test was used to compare EVNLP and control group spectral features. Differences were considered significantly different when P-value < 0.05. RESULTS: We detected over 13,000 spectral features of which 58 met the significance criteria with biologically relevant putative identifications. Furthermore we were able to confirm the identities of the ions taurine (P-value: 0.002) and tryptophan (P-value: 0.002), which were among the most significantly perturbed ions at 24 hours between the experimental and control groups. Metabolites belonging to the following pathways were the most perturbed at 24 hours: neuroactive ligand-receptor interaction (P-values: 0.031 and 0.036) and amino acid metabolism, including tyrosine and tryptophan metabolism (P-values: 0.015, 0.002, and 0.017). Taurine abundance decreased and tryptophan abundance increased at 24 hours. Other metabolites also identified at 24 hours included phenylalanine, xanthosine, and citric acid (P-values: 0.002, 0.002, and 0.0152). DISCUSSION: This study showed presence of active metabolism during EVNLP and metabolic derangement toward the end of perfusion, which correlated with detection of altered mitochondrial structure, swelling, and elongation.

Application of epineural sheath conduit for restoration of 6-cm long nerve defects in a sheep median nerve model.

BACKGROUND: Due to limited number of studies, we tested feasibility of autologous epineural sheath conduit (ESC) in repair of 6-cm median nerve gaps in a sheep-the large animal model. MATERIALS AND METHODS: Eight ewes, 6-8 months old, 30-35 kg, were divided into three experimental groups: group 1-no defect repair (n = 4 nerves/group), group 2-autograft controls (n = 6 nerves/group), group 3-autologous ESC filled with saline (n = 6 nerves/group). ESC was constructed from a 6-cm long segment of sheep median nerve and tested for expression of laminin B, Glial fibrillary acidic protein (GFAP), S-100 and CD31 using immunofluorescent staining. At 6 months after nerve repair, nerve conduction velocity and somatosensory evoked potentials (SSEP) assessed neurosensory recovery, while histomorphometry tested nerve regeneration. RESULTS: Ex vivo characterization of ESC, before in vivo nerve gap repair, showed high laminin B expression, which supports axonal growth. At 6 months post-repair, structural integrity of ESC was preserved. ESC was well-vascularized and tissue adhesions were comparable to autograft controls. The maximal conduction velocities (29.80 ± 5.85 ms vs. 32.28 ± 6.75 ms; p = .44), action potential amplitudes (32.68 ± 17.44 mV vs. 44.14 ± 23.10 mV; p = .38) and SSEP amplitude values (6.18 ± 5.84 mV vs. 4.68 ± 2.53 mV; p = .28) were comparable between autograft and ESC groups. Presence of regenerating axons was confirmed in the distal segment of ESC at 6 months after repair. CONCLUSION: The feasibility of ESC in restoration of 6-cm long nerve defects in a sheep median nerve model was confirmed by nerve conduction assessments and correlated with axonal regeneration tested by histomorphometry. We confirmed ESC potential in support of regeneration of long nerve defects.

Pyrimidine synthesis inhibition enhances cutaneous defenses against antibiotic resistant bacteria through activation of NOD2 signaling.

Multidrug-resistant bacterial strains are a rapidly emerging healthcare threat; therefore it is critical to develop new therapies to combat these organisms. Prior antibacterial strategies directly target pathogen growth or viability. Host-directed strategies to increase antimicrobial defenses may be an effective alternative to antibiotics and reduce development of resistant strains. In this study, we demonstrated the efficacy of a pyrimidine synthesis inhibitor, N-phosphonacetyl-L-aspartate (PALA), to enhance clearance of methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Acinetobacter baumannii strains by primary human dermal fibroblasts in vitro. PALA did not have a direct bactericidal effect, but enhanced cellular secretion of the antimicrobial peptides human ß-defensin 2 (HBD2) and HBD3 from fibroblasts. When tested in porcine and human skin explant models, a topical PALA formulation was efficacious to enhance MRSA, P. aeruginosa, and A. baumannii clearance. Topical PALA treatment of human skin explants also resulted in increased HBD2 and cathelicidin (LL-37) production. The antimicrobial actions of PALA required expression of nucleotide-binding, oligomerization domain 2 (NOD2), receptor-interacting serine/threonine-protein kinase 2 (RIP2), and carbamoyl phosphatase synthase II/aspartate transcarbamylase/dihydroorotase (CAD). Our results indicate that PALA may be a new option to combat multidrug-resistant bacterial infections of the skin through enhancement of an integral pathway of the cutaneous innate immune defense system.

Quantity of Lymph Nodes in the Vascularized Lymph Node Transfer Influences Its Lymphaticovenous Drainage.

BACKGROUND: The purpose of this study was to: (1) evaluate the mechanism of lymph drainage through a vascularized lymph node (VLN) flap, and (2) investigate if the number of VLNs impacts lymph transit time through the flap. METHODS: Twenty-seven axillary VLN flaps were elevated in 14 Sprague-Dawley rats and divided into three groups (n = 9 each) based on the number of lymph nodes present: group 1 (0-VLNs), group 2 (2-VLNs), and group 3 (4-VLNs). Indocyanine green (n = 8/group) and Alexa680-albumin (n = 1/group) were injected into the edge of flaps and the latency period between injection and fluorescence in the axillary vein was recorded. Stereomicroscopic fluorescent lymphography was performed to directly visualize lymphatic transit through VLNs. RESULTS: Fluorescence was detected in the axillary vein after 229s [47-476], 79s [15-289], and 56s [16-110] in group 1, 2, and 3, respectively (p < 0.01). There was a negative correlation between the number of VLNs in the flap and the latency period (r = -0.59; p < 0.05). Median flap weights were comparable in group 1, 2, and 3 (258 mg [196-349], 294 mg [212-407], 315 mg [204-386], respectively; p = 0.54). Stereoscopic lymphography allowed direct visualization of lymphatic fluid transit through VLNs. CONCLUSION: Lymphatic fluid in VLN flaps drains into the venous system mainly by passing through the afferent lymphatics and lymph nodes. A secondary mechanism appears to be the diffusion of fluid into the venous system via intratissue lymphaticovenous connections created during flap elevation. Increasing the number of lymph nodes in the flap is associated with a more rapid transit of fluid.

Developing a protocol for normothermic ex-situ limb perfusion.

BACKGROUND: Ischemia time represents a significant limitation for successful extremity transplantation because of the rapid deterioration of ischemic muscle. Normothermic ex-situ preservation is an emergent method to prolong the organ viability following procurement, by replicating the physiologic conditions. The aim of this study was to develop an ex-situ normothermic limb perfusion system to preserve the viability and function of porcine limbs for 12 hours following procurement. METHODS: A total of 18 swine limbs were perfused. Thirteen limbs were used to develop the perfusion protocol. Five limbs were perfused according to the optimized protocol. These limbs were perfused at 39°C for twelve hours using an oxygenated colloid solution containing red blood cells. Glucose and electrolytes were kept within physiologic range by partial perfusate exchange. Limb specific perfusion quality was assessed by muscle contractility upon electrical nerve stimulation, compartment pressure, creatine kinase (CK) and myoglobin concentrations, tissue oxygen saturation (near infrared spectroscopy), indocyanine green angiography, and infrared radiation by thermographic imaging. RESULTS: The last five limbs reached the 12 hours' perfusion target maintaining normal compartment pressure (16.4 ± 8.20 mmHg), minimal weight increase (0.54 ± 7.35%), and mean muscle temperature of 33.6 ± 1.67°C. Myoglobin and CK concentrations were 875 ± 291.4 ng/mL, and 53344 ± 14850.34 U/L, respectively, at the end of perfusion. Muscle contraction was present in all limbs until cessation of perfusion. Differences in uniformity and quality of distal perfusion were identified with thermography and angiography imaging at 12 hours of perfusion. CONCLUSIONS: Ex-situ normothermic limb perfusion preserves swine limb physiology and function for at least 12 hours.

Application of Epineural Sheath as a Novel Approach for Fat Volume Maintenance.

INTRODUCTION: Various methods have been suggested to improve fat graft survival and decrease graft loss. The exact mechanism of fat graft survival is still unclear, and new strategies are needed to further investigate it. MATERIALS AND METHODS: The efficacy of epineural sheath in fat volume maintenance was tested in rat model. Five experimental groups were created: group 1, fat graft without any coverage; group 2, epineural sheath tube alone; group 3, epineural sheath tube filled with fat graft; group 4, fat graft mixed with minced epineural sheath without any coverage; and group 5, fat graft covered with the epineural sheath patch. All grafts were implanted into the dorsal subcutaneous region and were followed for up to 12 weeks, when samples were harvested for hematoxylin and eosin and immunostaining for vascular endothelial growth factor expression and perilipin evaluation of fat viability. RESULTS: In groups 1 and 4, over 25% of graft loss was observed at first week, over 50% at third week, and 100% at sixth week postimplantation. The weight of fat graft within the epineural sheath tube and the weight of epineural tube (ET) alone were maintained up to 12 weeks postimplantation. The weight of fat graft within the epineural patch was maintained up to 6 weeks, but 50% of weight loss was observed between 6 and 12 weeks. Structure of the epineural sheath tubes and patches was intact, and no leakage of fat graft was observed. Based on hematoxylin and eosin staining, normal structure and integrity of the fat graft within the ET were preserved up to 12 weeks postimplantation. Characteristic adipocyte morphology was confirmed by perilipin staining, showing viable fat cells in groups 3 and 5 at 12 weeks. Increased vascular endothelial growth factor expression was observed in groups 2, 3, 4, and 5. CONCLUSIONS: Both, the ETs and epineural patches maintained 100% and 50% of fat graft weight at 12 weeks postimplantation, respectively. These results were confirmed by histology and immunostaining showing viable adipocytes within the epineural patches (6 weeks) and tubes (12 weeks). These results are encouraging and justify further evaluation of fat volume maintenance in preclinical large animal model in preparation to clinical application.

The reversed paradigm of chimerism induction: Donor conditioning with recipient-derived bone marrow cells as a novel approach for tolerance induction in vascularized composite allotransplantation.

PURPOSE: To test a new approach of donor conditioning with recipient bone marrow cells (BMC) to induce tolerance in vascularized composite allograft (VCA) transplantation. METHODS: Lewis rats' (recipients) BMC were stained with PKH-26. The ACI rats (donors) were conditioned with 80 × 106 Lewis BMC, 24 or 72 hours before VCA (groin flap) transplantation. Forty-eight VCA were performed between ACI donors and Lewis recipients. In groups I and II, donors were preconditioned (24 and 72 hours before transplantation, respectively), and recipients received 7-day anti-αß-TCR/cyclosporine-A post-transplantation. In groups III and IV, donors were preconditioned (24 and 72 hours before transplantation, respectively), and recipients received no systemic immunosuppression. In group V, recipients received 7-day anti-αß-TCR/cyclosporine-A post-transplantation. In group VI, recipients received no systemic immunosuppression. Assessment included evaluation of transplant viability and induction of donor-specific chimerism via flow cytometry, immunofluorescence, and PCR. RESULTS: Groups III, IV, and VI rejected allografts, at an average of 14 ± 5.2, 10 ± 2.7, and 8 ± 0.7 days. In groups I, II, and V, the mean survival was 80 ± 18.2 (p = 0.0002), 64 ± 27.4 (p = 0.001), and 30 ± 4.7 (p = 0.02) days. In groups I and II, donor-specific chimerism in the blood decreased from 8.8 ± 3.4% and 8.6 ± 3.4% on day 7 to 3.7 ± 1.32% (p = 0.02) and 4.7 ± 2.7% when the flaps manifested grade 3 rejection. The presence of PKH-26+ Lewis BMC was confirmed in the donor's blood, bone marrow, lymphoid organs, and liver (preconditioned at 24 and 72 hours). CONCLUSIONS: Donor preconditioning is a novel approach modifying recipient's responsiveness to donor allograft and prolonging the allograft survival under short-term immunosuppression. © 2015 Wiley Periodicals, Inc. Microsurgery 36:676-683, 2016.

Vascularized axillary lymph node transfer: A novel model in the rat.

Vascularized lymph node transfer (VLNT) is a promising microvascular free flap technique for the surgical treatment of lymphedema. To date, few experimental animal models for VLNT have been described and the viability of lymph nodes after the transfer tested. We aimed to evaluate the feasibility of axillary VLNT in the rat. Lymph node containing flaps were harvested from the axillary region in 10 Lewis rats based on the axillary vessels. Flaps were transferred to the ipsilateral groin and end-to-side microanastomosis was performed to the femoral vessels using 10-0 sutures. Indocyanine green (ICG) angiography was used to confirm flap perfusion. On postoperative day 7, flaps were elevated to assess their structure and vessel patency. Hematoxylin and eosin staining was used to confirm the presence and survival of lymph nodes. All animals tolerated the procedure well. Immediate post-procedure ICG angiography confirmed flap perfusion. No signs of ischemia or necrosis were observed in donor extremities. At postoperative day 7, all flaps remained viable with patent vascular pedicles. Gross examination and histology confirmed the presence of 3.6 ± 0.5 lymph nodes in each flap without any signs of necrosis. This study showed that the transfer of axillary lymph nodes based on the axillary vessels is feasible. The flap can be used without the need for donor animals and it contains a consistent number of lymph nodes. This reliable VLNT can be further utilized in studies involving lymphedema, transplantation, and induction of immunologic tolerance.

A new vascularized cervical lymph node transplantation model: an anatomic study in rats.

INTRODUCTION: Vascularized lymph node transfer is of high interest for the treatment of lymphedema. Currently, there are few experimental small animal models of vascularized lymph node transplantation. In this article, our aim was to describe a new vascularized cervical lymph node transplantation model in rats. MATERIALS AND METHODS: Ten male Sprague-Dawley rats weighing 200 to 250 g were used in this study. The anatomic features of the neck lymph nodes in rats were explored. Anatomic neck dissections were performed, and lymph node flaps were harvested. The common carotid artery and the jugular vein were used as the vascular pedicles of the lymph node flap. Methylene blue dye was injected into the arterial pedicle. Lymph nodes were identified, and their structure was confirmed by histological evaluation. Laser-assisted indocyanine green angiography was used to confirm perfusion of the lymph node flap. RESULTS: An adequate perfusion was observed in the lymph node flap. The dye disseminated evenly within the lymph nodes, indicating that the flap had a well-established vascular network and an adequate blood supply. Macroscopically, perfusion of 5 to 6 lymph nodes was observed. Histological examination of tissue samples confirmed well-defined lymph nodes. After indocyanine green administration, fluorescence was observed throughout the lymph node flap and within the venous pedicle of the flap. CONCLUSIONS: To the best of our knowledge, this is the first report describing vascularized lymph node flap in the head and neck region of a rat. Our lymph node flap preparation technique confirmed the presence of 5 to 6 lymph nodes within the flap. The presented vascularized lymph node flap can be applied to transplantation studies, lymphedema studies, and to studies on immunological mechanism of tolerance and rejection.

Repair of the peripheral nerve gap with epineural sheath conduit to prevent muscle denervation atrophy in the diabetic rat model.

UNLABELLED: Muscle denervation atrophy is a result of lower motor neuron injury, thus an early restitution of muscle stimulation is essential in prevention of atrophic changes. THE AIM OF THE STUDY: To evaluate the new application of naturally occurring epineural sheath conduit in repair of the peripheral nerve gap to prevent development of muscle denervation atrophy. MATERIAL AND METHODS: We used the model of 20 mm sciatic nerve gap, resulting in denervation atrophy of the gastrocnemius muscle in the diabetic rats (DM type 2, n=42, Zucker Diabetic Fatty strain). We applied the epineural sheath conduit created from the autologous sciatic nerve for gap repair. Muscle atrophy was assessed with the Gastrocnemius Muscle Index (GMI) and microscopic muscle morphometry (mean fiber area) at 6 and 12 postoperative week. Muscle regeneration in the experimental group was compared to the gold-standard technique of autologous nerve grafting for the repair of created nerve gap. RESULTS: The GMI evaluation revealed comparable muscle mass restoration in groups with nerve repair using both epineural sheath and standard autologous nerve grafting (reaching 28 and 35% of contralateral muscle mass at 12 postoperative week, respectively, p=0.1), and significantly better restoration when compared to the negative control group (no repair, 20%, p<0.01). Micromorphometry confirmed significantly larger area of the regenerated muscle fibers in groups with both nerve grafting and epineural sheath conduit repair (reaching for both ca. 42% of the non-operated side), when compared to severe atrophic outcome when no nerve repair was performed (14% of the control fiber area, p<0.0001). The effectiveness of epineural conduit technique in muscle mass restoration was observed between 6 and 12 weeks after nerve repair--when gastrocnemius muscle mass increased by 12%. CONCLUSIONS: Peripheral nerve gap repair with naturally occurring epineural sheath conduit is effective in prevention of muscle denervation atrophy. This method is applicable in diabetic model conditions, showing results of regeneration which are comparable to the autologous nerve graft repair.

Effectiveness of topical immunosuppressants in prevention and treatment of rejection in face allotransplantation.

BACKGROUND: The use of topical immunosuppressants has been anecdotally reported for the treatment of rejection in vascularized composite allotransplantation. The aim of this study was to evaluate the effectiveness of topical tacrolimus and clobetasol in the prevention and treatment of rejection. METHODS: Seventy-six hemiface allotransplants, between ACI (RT1) donors and Lewis (RT1) recipients, were performed in 11 groups and treated with topical tacrolimus or clobetasol, or in combination with systemic cyclosporine A and anti-αß-T-cell receptor antibody for 1 week. Topical treatment increased the survival of the allograft in all groups. RESULTS: Best outcomes were obtained in the groups treated with systemic therapy and topical tacrolimus. Expression of proinflammatory cytokines interleukin 2, interferon γ, tumor necrosis factor α, and transforming growth factor ß correlated with clinical signs of rejection and the final outcomes. Clobetasol application was associated with a marked depletion of lymphocytic populations, and dermal and epidermal atrophy. CONCLUSIONS: Both topical tacrolimus and clobetasol were effective in treating episodes of acute rejection, and the best outcomes were achieved when their application was initiated after systemic immunosuppression. Topical tacrolimus proved to be a preferable adjunct agent to the systemic therapy by preventing both the local and systemic complications.

Natural conduits for bridging a 15-mm nerve defect: comparison of the vein supported by muscle and bone marrow stromal cells with a nerve autograft.

OBJECT: The gold standard for reconstructing large nerve defects, the nerve autograft, results in donor-site morbidity. This detrimental consequence drives the search for alternatives. We used a vein filled with a small piece of fresh muscle to prevent the vein from collapsing and with bone marrow stromal cells (BMSCs) to enhance regeneration. METHODS: In 60 rats, a 15-mm sciatic nerve defect was bridged with a nerve autograft, a vein filled with muscle or a vein filled with muscle and BMSCs. Toe spread and pinprick were used to evaluate motor and sensory function. Compound muscle action potentials (CMAPs) and the gastrocnemius muscle index (GMI) were recorded to assess conduction properties and denervation atrophy. Extensive histology was performed to confirm presence of BMSCs and to evaluate regeneration by staining neural tissue for Schwann cells and neural growth factor. RESULTS: After 12 weeks, all animals responded with toe spread and pinprick reaction; significant differences were found between groups. Six weeks post grafting no difference was found comparing the GMI between the groups. Group I had a significant increase in GMI at 12 weeks compared to group II and group III. The CMAP measurements showed comparable results at 6 weeks post grafting. Twelve weeks after reconstruction, group I had significantly better results compared to group II and group III. Group III showed a tendency to outperform group II at 12 weeks postoperatively. Immunofluorescence analysis showed an increased number of Schwann cells in group III compared to group II. The BMSCs were visible 6 and 12 weeks postoperatively. CONCLUSIONS: This study is a step forward in the search for an alternative to the nerve autograft because it demonstrates the beneficial effect of BMSCs to a conduit. However, our data do not demonstrate sufficient benefit to warrant clinical implementation at this stage.

Application of cell-based therapies in facial transplantation.

The main purpose of cellular therapy application in face transplantation is the continuous need for the development of new strategies that would eliminate the use of toxic immunosuppressive protocols. Cellular therapy in transplantation can significantly benefit allograft survival and shorten healing time. Cells used for a therapeutic purpose are isolated mostly from bone marrow and adipose tissues. They have the ability to proliferate and differentiate in the transplanted tissue and have immunomodulatory activity. Most of the cellular therapies such as T-regulatory, dendritic, and chimeric cells are still in the experimental stage. Molecular characterization of these cells and the mechanism of their participation in allograft acceptance and rejection are not well established and will contribute to the future of modern transplantology.

Augmented osteolysis in SPARC-deficient mice with bone-residing prostate cancer.

Prostate cancer preferentially metastasizes to bone, which is rich in structural and matricellular proteins capable of altering prostate cancer progression. This study explores the role of the bone stromal matricellular protein SPARC (osteonectin/BM-40) in the progression of bone metastatic prostate cancer. Quantification of bone destruction analyzed by micro-computed tomography showed augmented osteoclastic resorption, characterized by decreases in several morphometric bone parameters in SPARC knock out (KO) tibiae harboring RM1 murine prostate cancer cells compared with wild type (WT) animals. Tumor progression stimulated osteoclast formation, which was augmented in SPARC KO mice. In vitro differentiation of SPARC KO osteoclasts indicated accelerated progenitor expansion and formation of tartrate-resistant acid phosphatase-positive osteoclast-like cells with increased resorptive capacity, a mechanism resulting in enhanced tumor-induced bone loss in vivo. Whereas altered bone structure due to SPARC KO played a role in increased osteolysis, the enhanced osteolysis was primarily the result of increased resorption by SPARC KO osteoclasts. Our findings indicate that bone stromal SPARC suppresses tumor-induced bone lesion expansion by limiting osteoclast maturation and function.

A novel role for platelet secretion in angiogenesis: mediating bone marrow-derived cell mobilization and homing.

Angiogenesis alleviates hypoxic stress in ischemic tissues or during tumor progression. In addition to endothelial cell proliferation and migration, the angiogenic process requires bone marrow-derived cell (BMDC) recruitment to sites of neovascularization. However, the mechanism of communication between hypoxic tissues and the BM remains unknown. Using 2 models of hypoxia-induced angiogenesis (ischemic hindlimb surgery and subcutaneous tumor growth), we show that platelet infusion promotes BMDC mobilization into the circulation, BMDC recruitment into growing neovasculature, tumor vascularization, and blood flow restoration in ischemic limbs, whereas platelet depletion inhibits these effects. Thus, platelets are required for BMDC recruitment into ischemia-induced vasculature. Secretion of platelet α-granules, but neither dense granules nor platelet aggregation is crucial for BMDC homing and subsequent angiogenesis, as determined using VAMP-8(-/-), Pearl, and integrin Beta 3(-/-) platelets. Finally, platelets sequester tumor-derived promoters of angiogenesis and BMDC mobilization, which are counterbalanced by the antiangiogenic factor thrombospondin-1. A lack of thrombospondin-1 in platelets leads to an imbalance in proangiogenic and antiangiogenic factors and accelerates tumor growth and vascularization. Our data demonstrate that platelets stimulate BMDC homing in a VAMP-8-dependent manner, revealing a previously unknown role for platelets as key mediators between hypoxic tissues and the bone marrow during angiogenesis.