Tacrolimus-Eluting Disk within the Allograft Enables Vascularized Composite Allograft Survival with Site-Specific Immunosuppression without Systemic Toxicity.

AIM: Widespread clinical application of vascularized composite allotransplantation (VCA) has been limited by the need for lifelong systemic immunosuppression to prevent rejection. Our goal was to develop a site-specific immunosuppressive strategy that promotes VCA allograft survival and minimizes the risk of systemic side effects. METHODS: Tacrolimus loaded polycaprolactone (TAC-PCL) disks were prepared and tested for their efficacy in sustaining VCA allograft survival via site-specific immunosuppression. Brown Norway-to-Lewis rat hind limb transplantations were performed; animals received one TAC disk either in the transplanted (DTx) or in the contralateral non-transplanted (DnonTx) limbs. In another group, animals received DTx and lymphadenectomy on Tx side. Blood and allograft levels of TAC were measured using LC-MS/MS. Systemic toxicity was evaluated. RESULTS: Animals that received DTx achieved long-term allograft survival (> 200 days) without signs of metabolic and infectious complications. In these animals, TAC blood levels were low but stable between 2 to 5 ng/mL for nearly 100 days. High concentrations of TAC were achieved in the allografts and the draining lymph nodes (DLN). Animals that underwent lymphadenectomy rejected their allograft by 175 days. Animals that received DnonTx rejected their allografts by day 70. CONCLUSION: Controlled delivery of TAC directly within the allograft (with a single TAC disk) effectively inhibits rejection and prolongs VCA allograft survival, while mitigating the complications of systemic immunosuppression. There was a survival benefit of delivering TAC within the allograft as compared to a remote site. We believe this approach of local drug delivery has significant implications for drug administration in transplantation.

Treg-inducing microparticles promote donor-specific tolerance in experimental vascularized composite allotransplantation.

For individuals who sustain devastating composite tissue loss, vascularized composite allotransplantation (VCA; e.g., hand and face transplantation) has the potential to restore appearance and function of the damaged tissues. As with solid organ transplantation, however, rejection must be controlled by multidrug systemic immunosuppression with substantial side effects. As an alternative therapeutic approach inspired by natural mechanisms the body uses to control inflammation, we developed a system to enrich regulatory T cells (Tregs) in an allograft. Microparticles were engineered to sustainably release TGF-ß1, IL-2, and rapamycin, to induce Treg differentiation from naïve T cells. In a rat hindlimb VCA model, local administration of this Treg-inducing system, referred to as TRI-MP, prolonged allograft survival indefinitely without long-term systemic immunosuppression. TRI-MP treatment reduced expression of inflammatory mediators and enhanced expression of Treg-associated cytokines in allograft tissue. TRI-MP also enriched Treg and reduced inflammatory Th1 populations in allograft draining lymph nodes. This local immunotherapy imparted systemic donor-specific tolerance in otherwise immunocompetent rats, as evidenced by acceptance of secondary skin grafts from the hindlimb donor strain and rejection of skin grafts from a third-party donor strain. Ultimately, this therapeutic approach may reduce, or even eliminate, the need for systemic immunosuppression in VCA or solid organ transplantation.

Low-Cost, Three-Dimensionally-Printed, Anatomical Models for Optimization of Orbital Wall Reconstruction.

BACKGROUND: Orbital blowout fracture reconstruction often requires an implant, which must be shaped at the time of surgical intervention. This process is time-consuming and requires multiple placement trials, possibly risking complications. Three-dimensional printing technology has enabled health care facilities to generate custom anatomical models to which implants can be molded to precisely match orbital anatomy. The authors present their early experience with these models and their use in optimizing orbital fracture fixation. METHODS: Maxillofacial computed tomographic scans from patients with orbital floor or wall fractures were prospectively obtained and digitally reconstructed. Both injured-side and mirrored unaffected-side models were produced in-house by stereolithography printing technique. Models were used as templates for molding titanium reconstruction plates, and plates were implanted to reconstruct the patients' orbital walls. RESULTS: Nine patients (mean age, 15.5 years) were included. Enophthalmos was present in seven patients preoperatively and resolved in six patients with surgery. All patients had excellent conformation of the implant to the fracture site on postoperative computed tomographic scan. Postoperative fracture-side orbital volumes were significantly less than preoperative, and not significantly different from unfractured-side orbital volumes. Total model preparation time was approximately 10 hours. Materials cost was at most $21. Plate bending time was approximately 60 seconds. CONCLUSIONS: Patient-specific orbital models can speed the shaping of orbital reconstruction implants and potentially improve surgical correction of orbital fractures. Production of these models with consumer-grade technology confers the same advantages as commercial production at a fraction of the cost and time. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Long-term survival of limb allografts induced by pharmacologically conditioned, donor alloantigen-pulsed dendritic cells without maintenance immunosuppression.

BACKGROUND: We showed recently that limb allograft survival could be enhanced by administration of alloantigen (Ag)-pulsed immature dendritic cells (DC) after transplantation. Since indefinite graft survival was not achieved, we have further modified the DC by pharmacologic (rapamycin; Rapa) conditioning and ascertained their influence on graft survival, without continued immunosuppressive therapy. METHODS: We compared the ability of donor Ag-pulsed, Rapa-conditioned rat myeloid DC (Rapa DC) and control DC (CTR DC) to inhibit alloreactive T-cell responses after limb transplantation in antilymphocyte serum (ALS)-treated recipients given a short postoperative course of cyclosporine (CsA). RESULTS: Both DC populations expressed similar levels of major histocompatibility complex (MHC) II, CD40 and CD54, but Rapa DC expressed lower CD86. After toll-like receptor activation, both populations produced minimal interleukin (IL)-12p70, but Rapa DC secreted lower levels of IL-6 and IL-10. The capacity of DCs to stimulate T-cell proliferation in mixed leukocyte reactions was very low. Pulsing of the DC with donor Ag did not alter their phenotype or function. Interestingly, posttransplant administration of donor Ag-pulsed Rapa DC to rats given perioperative ALS and 21 days CsA significantly delayed graft rejection and promoted long-term (>125 days) graft survival. AlloAg-pulsed Rapa DC induced T-cell hyporesponsiveness and promoted the generation of IL-10-secreting CD4 T cells upon ex vivo challenge. CONCLUSIONS: Infusion of donor Ag-pulsed, Rapa-conditioned DC after composite tissue transplantation can prevent rejection of the grafts, including skin, across a full MHC mismatch and in the absence of continued immunosuppressive therapy.

Lateral Branch of the Thoracodorsal Nerve (LaT Branch) Transfer for Biceps Reinnervation.

In cases of significant upper extremity trauma, the thoracodorsal nerve is a reliable secondary option for the restoration of elbow flexion. In all previous descriptions, however, the entire nerve is transferred. We describe a case utilizing the lateral thoracodorsal nerve (LaT) branch for biceps reinnervation with an associated cadaver study. Transfer of the LaT branch to the biceps branch was performed on a patient who had sustained a traumatic brachial plexus injury that left him without elbow flexion. Also, 4 cadavers (8 upper extremities) were dissected to identify the bifurcation of the thoracodorsal nerve and confirm the feasibility of transferring the LaT branch to the biceps motor branch. Axon counts of the thoracodorsal proper, LaT branch, musculocutaneous proper, and the biceps branch were also obtained. A bifurcation of the thoracodorsal nerve was present in all cadaver specimens, with an average distance of 7.5 cm (range, 6.2-9.8 cm) from the insertion of the latissimus dorsi muscle. Axon counts revealed a donor-to-recipient ratio of 0.85:1. Follow-up of our patient at 1 year showed improvement of elbow flexion manual muscle testing grade from 0 to 4/5. Furthermore, electromyography at 1 year confirmed biceps reinnervation and showed normal readings of the latissimus compared with preoperative electromyography. Transfer of the LaT branch is a viable and minimally morbid option for biceps reinnervation after traumatic branchial plexus injury. Further follow-up of our patient and larger prospective studies are needed to understand the true potential of this nerve transfer.

Totally Implantable Wireless Ultrasonic Doppler Blood Flowmeters: Toward Accurate Miniaturized Chronic Monitors.

Totally implantable wireless ultrasonic blood flowmeters provide direct-access chronic vessel monitoring in hard-to-reach places without using wired bedside monitors or imaging equipment. Although wireless implantable Doppler devices are accurate for most applications, device size and implant lifetime remain vastly underdeveloped. We review past and current approaches to miniaturization and implant lifetime extension for wireless implantable Doppler devices and propose approaches to reduce device size and maximize implant lifetime for the next generation of devices. Additionally, we review current and past approaches to accurate blood flow measurements. This review points toward relying on increased levels of monolithic customization and integration to reduce size. Meanwhile, recommendations to maximize implant lifetime should include alternative sources of power, such as transcutaneous wireless power, that stand to extend lifetime indefinitely. Coupling together the results will pave the way for ultra-miniaturized totally implantable wireless blood flow monitors for truly chronic implantation.

Single Implantable FK506 Disk Prevents Rejection in Vascularized Composite Allotransplantation.

BACKGROUND: In vascularized composite allotransplantation, medication nonadherence leads to increased acute rejections. Improving medication adherence would improve overall allograft survival. Regionally delivered immunosuppression, targeted to sites of allorecognition, may reduce or eliminate the need for daily systemic immunosuppression. METHODS: The authors developed biodegradable FK disks containing FK506-loaded double-walled microspheres and tested their efficacy at preventing rejection in a Brown-Norway-to-Lewis rat hindlimb transplantation model. In some experimental group animals, one FK disk was implanted subcutaneously either in native nontransplanted leg or in a transplanted allograft. Regular blood FK506 levels were measured. The endpoint was 180-day allograft survival or grade 3 rejection. At the endpoint, tissue FK506 levels were measured and mixed lymphocytic reaction was performed. RESULTS: A single FK disk maintained systemic blood FK506 levels between 5 and 15 ng/ml for 146 ± 11.1 days. After that, the levels declined to less than 5 ng/ml through the endpoint. There was significantly increased FK506 concentration in groin lymph nodes draining the implanted FK disk. Compared with other groups, animals with an FK disk in the transplanted allograft had 100 percent allograft survival to more than 180 days despite subtherapeutic levels below 5 ng/ml. In these animals, significant T-cell hyporesponsiveness was seen in groin lymph nodes draining the FK disk compared with robust splenic T-cell proliferation. CONCLUSIONS: Sustained regional immunosuppression (with a single FK506 disk) maintained the allograft by means of a high regional concentration of FK506. Notably, this was achieved at subtherapeutic blood concentrations of FK506, without any further systemic FK506 administration.

A System for Simple Real-Time Anastomotic Failure Detection and Wireless Blood Flow Monitoring in the Lower Limbs.

Current totally implantable wireless blood flow monitors are large and cannot operate alongside nearby monitors. To alleviate the problems with the current monitors, we developed a system to monitor blood flow wirelessly, with a simple and easily interpretable real-time output. To the best of our knowledge, the implanted electronics are the smallest in reported literature, which reduces bio-burden. Calibration was performed across realistic physiological flow ranges using a syringe pump. The device's sensors connected directly to the bilateral femoral veins of swine. For each 1 min, blood flow was monitored, then, an occlusion was introduced, and then, the occlusion was removed to resume flow. Each vein of four pigs was monitored four times, totaling 32 data collections. The implant measured 1.70 cm3 without battery/encapsulation. Across its calibrated range, including equipment tolerances, the relative error is less than ±5% above 8 mL/min and between -0.8% and +1.2% at its largest calibrated flow rate, which to the best of our knowledge is the lowest reported in the literature across the measured calibration range. The average standard deviation of the flow waveform amplitude was three times greater than that of no-flow. Establishing the relative amplitude for the flow and no-flow waveforms was found necessary, particularly for noise modulated Doppler signals. Its size and accuracy, compared with other microcontroller-equipped totally implantable monitors, make it a good candidate for future tether-free free flap monitoring studies.

IGF-1 and Chondroitinase ABC Augment Nerve Regeneration after Vascularized Composite Limb Allotransplantation.

Impaired nerve regeneration and inadequate recovery of motor and sensory function following peripheral nerve repair remain the most significant hurdles to optimal functional and quality of life outcomes in vascularized tissue allotransplantation (VCA). Neurotherapeutics such as Insulin-like Growth Factor-1 (IGF-1) and chondroitinase ABC (CH) have shown promise in augmenting or accelerating nerve regeneration in experimental models and may have potential in VCA. The aim of this study was to evaluate the efficacy of low dose IGF-1, CH or their combination (IGF-1+CH) on nerve regeneration following VCA. We used an allogeneic rat hind limb VCA model maintained on low-dose FK506 (tacrolimus) therapy to prevent rejection. Experimental animals received neurotherapeutics administered intra-operatively as multiple intraneural injections. The IGF-1 and IGF-1+CH groups received daily IGF-1 (intramuscular and intraneural injections). Histomorphometry and immunohistochemistry were used to evaluate outcomes at five weeks. Overall, compared to controls, all experimental groups showed improvements in nerve and muscle (gastrocnemius) histomorphometry. The IGF-1 group demonstrated superior distal regeneration as confirmed by Schwann cell (SC) immunohistochemistry as well as some degree of extrafascicular regeneration. IGF-1 and CH effectively promote nerve regeneration after VCA as confirmed by histomorphometric and immunohistochemical outcomes.

The Development of a Wireless Implantable Blood Flow Monitor.

Microvascular anastomotic failure remains an uncommon but devastating problem. Although the implantable Doppler probe is helpful in flap monitoring, the devices are cumbersome, easily dislodged, and plagued by false-positive results. The authors have developed an implantable wireless Doppler monitor prototype from off-the-shelf components and tested it in a swine model. The wireless probe successfully distinguished between femoral vein flow, occlusion, and reflow, and wirelessly reported the different signals reliably. This is the first description of a wireless implantable blood flow sensor for flap monitoring. Future iterations will incorporate an integrated microchip-based Doppler system that will decrease the size to 1 mm, small enough to fit onto an anastomotic coupler.

Functional outcomes following multiple acute rejections in experimental vascularized composite allotransplantation.

BACKGROUND: Vascularized composite allotransplantation has become a clinical reality. Patients undergoing vascularized composite allotransplantation have modest functional return. Most patients have had multiple acute rejections. The effect of multiple acute rejections influencing functional outcomes is unknown. This study systematically analyzes the effects of multiple acute rejections on functional outcome. METHODS: Rat functional orthotopic hind-limb transplants were performed from Brown-Norway to Lewis rats. Group 1 consisted of isografts. In group 2, daily cyclosporine was administered to prevent acute rejection. In group 3, recipients did not receive regular immunosuppression but received only pulsed cyclosporine and dexamethasone to rescue acute rejection. The study endpoint was 90 days. Muscle and sciatic nerve biopsy specimens were taken for histologic analyses. Hind-limb function was assessed using sciatic nerve axon density, nerve conduction velocity, and muscle force generated by the gastrocnemius muscle. Novel video kinematics was used to analyze gait. RESULTS: By the endpoint, group 3 animals had 17 ± 5.1 acute rejections. Muscle biopsy showed significant atrophy and fibrosis in group 3 compared with groups 1 and 2. Withdrawal to pin prick was evident by days 31 ± 1.2, 30 ± 2.3, and 31 ± 3.7 in groups 1, 2, and 3, respectively. At the endpoint, there was no significant difference in the axon density or nerve conduction velocity among the three groups, but muscle force generated was significantly less in group 3. Gait was abnormal in group 3 animals compared with other groups. CONCLUSIONS: In this study, multiple acute rejections induced muscle atrophy and fibrosis and consequent decreased function. This emphasizes the importance of preventing acute rejection to achieve optimum function following vascularized composite allotransplantation.

A model for functional recovery and cortical reintegration after hemifacial composite tissue allotransplantation.

BACKGROUND: The ability to achieve optimal functional recovery is important in both face and hand transplantation. The purpose of this study was to develop a functional rat hemifacial transplant model optimal for studying both functional outcome and cortical reintegration in composite tissue allotransplantation. METHODS: Five syngeneic transplants with motor and sensory nerve appositions (group 1) and five syngeneic transplants without nerve appositions (group 2) were performed. Five allogeneic transplants were performed with motor and sensory nerve appositions (group 3). Lewis (RT1) rats were used for syngeneic transplants and Brown-Norway (RT1) donors and Lewis (RT1) recipients were used for allogeneic transplants. Allografts received cyclosporine A monotherapy. Functional recovery was assessed by recordings of nerve conduction velocity and cortical neural activity evoked by facial nerve and sensory (tactile) stimuli, respectively. RESULTS: All animals in groups 1 and 3 showed evidence of motor function return on nerve conduction testing, whereas animals in group 2, which did not have nerve appositions, did not show electrical activity on electromyographic analysis (p < 0.001). All animals in groups 1 and 3 showed evidence of reafferentation on recording from the somatosensory cortex after whisker stimulation. Animals in group 2 did not show a cortical response on stimulation of the whiskers (p < 0.001). CONCLUSION: The authors have established a hemiface transplant model in the rat that has several modalities for the comprehensive study of motor and sensory recovery and cortical reintegration after composite tissue allotransplantation.

Daily topical tacrolimus therapy prevents skin rejection in a rodent hind limb allograft model.

BACKGROUND: Skin is the most immunogenic component of a composite tissue allograft. Topical immunotherapy is an attractive therapeutic modality with which to provide local immunosuppression, with minimal systemic toxicity. The present study was performed to investigate the potential of topical tacrolimus to prolong survival of the skin component of a composite tissue allograft. METHODS: Wistar Furth-to-Lewis rat orthotopic hind limb transplants were performed. Group I consisted of rats treated with topical tacrolimus; group II, antilymphocyte serum plus 21 days cyclosporine; and group III, antilymphocyte serum plus 21 days of cyclosporine plus topical tacrolimus. In group IV, tacrolimus levels in blood, skin, and muscle were measured in an autograft control group. RESULTS: All animals in group I (n = 8) developed grade III clinical rejection by postoperative day 9. In group II (n = 9), the median onset of grade III rejection was postoperative day 40 (range, postoperative days 34 to 44). In group III (n = 6), two animals developed focal grade III rejection on postoperative days 35 and 56. The remaining four animals reached the 100-day endpoint without grade III rejection. In group IV, tacrolimus levels were low or undetectable in blood, whereas skin levels were 100-fold higher than underlying muscle. CONCLUSIONS: Topical tacrolimus therapy has the potential to prevent skin rejection in a composite tissue allograft. Preoperative depletion of T cells with antilymphocyte serum, along with a short course of systemic immunosuppression, prevents acute rejection, whereas topical tacrolimus inhibits immune cell function in the skin. Concentrations of tacrolimus are substantially higher in skin compared with underlying muscle and peripheral blood. Topical immunotherapy could reduce the morbidity associated with systemic immunosuppression in clinical composite tissue allografts.

Allopeptide-pulsed dendritic cells and composite tissue allograft survival.

Composite tissue allografts (CTAs) contain their own reservoir of vascularized bone marrow, offering novel aspects for the induction of donor-specific tolerance. Additionally, the manipulation of recipient dendritic cells, pulsed with donor allopeptide, has been shown to engender solid organ allograft survival. To exploit these modalities, we have developed a protocol utilizing injection of recipient bone marrow-derived dendritic cells (BMDCs) pulsed with a donor-derived peptide for use in CTA transplantation. Six days prior to orthotopic hind-limb transplantation, Lewis rats received IV injection of donor allopeptide-pulsed, recipient BMDCs, in conjunction with a single dose of anti-lymphocyte serum. Control groups displayed signs of allograft rejection within 5 days postoperatively. Animals within the primary experimental cohort demonstrated prolongation of graft survival to an average of 8 days, and exhibited low numbers of donor T cells. The use of BMDCs in conjunction with transient immunosuppression has potential therapeutic application for induction of donor-antigen-specific tolerance to hind limb allografts.

Intrinsic ability of GM+IL-4 but not Flt3L-induced rat dendritic cells to promote allogeneic T cell hyporesponsiveness.

The influence of GM+IL-4 and Flt3 ligand (FL) on phenotype and function of BM-derived DC from Lewis rats was investigated. GM+IL-4-induced DC, despite expression of CD80/CD86, were less stimulatory than FL-induced DC that expressed low CD80/CD86 and were efficient stimulators of allogeneic T cells. GM+IL-4 DC were CD11b+ OX62lo, whereas FL DC were CD11blo OX62+. Following activation, GM+IL-4 DC produced IL-10 and IL-6, but no IL-12p70, and were resistant to further maturation. FL DC produced IL-12p70, IFN-alpha/beta, IL-10 and IL-6 and underwent maturation. Repeated stimulation of T cells with GM+IL-4 DC inhibited proliferation, cytokine production and induced early T cell apoptosis. FL DC-activated T cells produced large amounts of IFN-gamma/IL-10 and exhibited late T cell apoptosis/necrosis. In vivo, GM+IL-4 DC induced alloAg-specific hyporesponsiveness following T cell restimulation. These results demonstrate that GM+IL-4 DC display intrinsic regulatory properties, inducing passive-cell-death in T cells with potential for inactivation/regulation of alloreactive T cells in transplantation.