The Banff 2007 working classification of skin-containing composite tissue allograft pathology.

Composite tissue allotransplantation (CTA) is a recently introduced option for limb replacement and reconstruction of tissue defects. As with other allografts, CTA can undergo immune-mediated rejection; therefore standardized criteria are required for characterizing and reporting severity and types of rejection. This article documents the conclusions of a symposium on CTA rejection held at the Ninth Banff Conference on Allograft Pathology in La-Coruna, Spain, on 26 June 2007, and proposes a working classification, the Banff CTA-07, for the categorization of CTA rejection. This classification was derived from a consensus discussion session attended by the first authors of three published classification systems, pathologists and researchers from international centers where clinical CTA has been performed. It was open to all attendees to the Banff conference. To the extent possible, the format followed the established National Institutes of Health (NIH) guidelines on Consensus Development Programs. By consensus, the defining features to diagnose acute skin rejection include inflammatory cell infiltration with involvement of epidermis and/or adnexal structures, epithelial apoptosis, dyskeratosis and necrosis. Five grades of severity of rejection are defined. This classification refines proposed schemas, represents international consensus on this topic, and establishes a working collective classification system for CTA reporting of rejection in skin-containing CTAs.

A method of endoscopic endotracheal intubation in rabbits.

Endotracheal intubation in rabbits is a formidable task and has led to the devising of a new endoscopic method. Sixty New Zealand White rabbits (2.0-3.5 kg) underwent experimental left thoracotomy requiring mechanical ventilation. After anaesthetic induction was achieved, the animal was placed in a supine position. A rigid 30 degrees endoscope was passed through a 4.5 mm endotracheal tube (ET) and then used to advance, under direct vision, through the oropharynx and vocal cords. Once passing the vocal cords, the ET was advanced into the trachea as the endoscope was withdrawn. All animals were successfully intubated within 30 s to 2 min and then ventilated. No complications or deaths occurred during or after intubation, or postoperatively. No instances of oesophageal intubation occurred. All animals survived long-term. Thirty animals underwent necropsy at 10 days and, 30 at 30 days, with no postoperative evidence of orotracheal injury. We have established a new method of endotracheal intubation in rabbits. It is reliable, effective, non-traumatic, safe, and expeditious.

BioGlue surgical adhesive for thoracic aortic repair during coagulopathy: efficacy and histopathology.

BACKGROUND: We hypothesized that induction of coagulopathy in sheep would model clinical needle hole and surgical bleeding from synthetic graft anastomoses, and that a new tissue bioadhesive (BioGlue) would control postoperative blood loss during surgical repair of the thoracic aorta. METHODS: Sheep were anticoagulated with aspirin and heparin. A bypass was made using end-to-side anastomoses of a graft to a partially occluded descending thoracic aorta. Experimental anastomoses (EXP, n = 9) were treated with BioGlue, and control anastomoses (CON, n = 5) were treated with Surgicel to gain intraoperative hemostasis. RESULTS: EXP animals exhibited significantly reduced postsurgical bleeding (CON median 955 mL versus EXP median 470 mL, p < 0.003), a reduced rate of blood loss over the first 2 postoperative hours (CON median 210 mL/hr versus EXP median 92.5 mL/hr, p < 0.006), and over the entire recovery period (CON median 158 mL/hr versus EXP median 86 mL/hr, p < 0.05), and reduced total blood loss (CON mean 1,497 +/- 691 mL versus EXP mean 668 +/- 285 mL, p < 0.008). On histologic examination of tissues explanted after 3 months, BioGlue was relatively inert and demonstrated a minimal inflammatory response. CONCLUSIONS: The use of BioGlue significantly reduced the volume and rate of postsurgical bleeding in a coagulopathic sheep model for thoracic aortic operations. Histopathologically, BioGlue generated only a minimal inflammatory response. This new surgical tissue bioadhesive should prove extremely beneficial for coagulopathic patients undergoing thoracic aortic or vascular procedures.

A novel technique in a sheep model for evaluating prosthetic heart valve performance.

There have been many various animal studies to evaluate the structural integrity and antithrombogenicity of prosthetic heart valves. We were interested in developing a novel sheep model to study the thrombogenicity of mechanical heart valves placed into the systemic circulation but without the need for cardiac bypass. Also, we wanted to minimize the risk ofparaplegia from complete thoracic aortic clamping. Six sheep underwent left lateral thoracotomy for placement of a mechanical heart valve in parallel with the descending thoracic aorta. A valved conduit with a dacron tube graft sutured to the back end was fashioned. Employing partial aortic occlusion with a side-biting clamp, the proximal and distal ends were anastomosed in an end-to-side fashion. Once flow was confirmed through the graft, the native aorta was occulded with umbilical tape. The sheep received no postoperative anticoagulation. The median operative time and estimated blood loss (EBL) was 170 min and 250 cc, respectively. Patency of the valved conduits was confirmed during the initial procedure, and there was no incidence of paraplegia postoperatively. Two animals expired shortly after extubation and at necropsy the valved conduits were patent with preserved valve function. The four survivors were sacrificed a median of 37 days postoperatively. Prior to euthanasia, the valved conduits were evaluated in situ with ultrasound. In all cases, the valves had clot formation at the hinges, which prevented active movement of the leaflets. This novel in vivo technique provides an alternative in testing the thrombogenicity of prosthetic heart valves without cardiac bypass or the risk of paraplegia in an animal that is extremely sensitive to complete aortic cross-clamp.

Recellularization of heart valve grafts by a process of adaptive remodeling.

The objective of this study was to investigate if function and durability of connective tissue grafts stems from in vivo revascularization and recellularization. Viability is important for durable valve performance, demonstrated by pulmonary autografts. A pattern of in vivo recellularization occurs in xenogeneic or allogeneic heart valves decellularized prior to implantation, dictated by the tissue matrix and functional biomechanics. Porcine or sheep heart valves were decellularized with the SynerGraft antigen reduction process (a common treatment process to remove all histologically demonstrable leaflet cells), and implanted as pulmonary (n = 11) or aortic valve (n = 9) replacements in sheep. Sheep allograft pulmonary valves (n = 4) were implanted as pulmonary valve replacements. Recellularization was evaluated histologically after 3, 4, 5, 6, and 11 months, with cell phenotypes identified using specific antibodies. SynerGraft heart valves were progressively recellularized beginning with an initial cellular infiltrate, and subsequent repopulation with mature interstitial cells. This process occurs in the conduit and then in the leaflet, and is associated with revascularization of the graft. Functional, fully developed fibrocytes, actively synthesizing type I procollagen (antibody probe) were present within 3 months. As the process matured cell density and distribution became similar to native valve leaflets with localization of smooth muscle actin positive cells at the ventricularis/spongiosa interface. After 11 months, leaflet explants had no detectable inflammatory cells, were as much as 80% repopulated, and had a distribution of smooth muscle actin positive cells similar to that of the natural leaflet. SynerGraft- treated heart valve implants are repopulated by a process typical of adaptive remodeling following implantation. This antigen reduction treatment is the first successful tissue engineering effort obtaining an implant with mature recipient cells capable of matrix protein synthesis. Normal early valve function and durability is maintained.

Multiple oblique illumination and high-definition microscopy for breast fine-needle aspirates.

The ability of multiple oblique illumination (MOI) and high-definition microscopy (Edge R-400 3-D microscope) to improve resolution of cellular detail in the evaluation of cytopathological specimens of Pap smears and thyroid fine-needle aspirates (FNAs) has been demonstrated. However, previous experiments showed that the advantages of MOI and high-definition stereo microscopy were less certain for the breast FNAs. We hypothesized that these findings were due to the lack of sample thickness for the breast FNA specimens. To test this hypothesis, we analyzed breast FNA specimens that were significantly thicker (10.5 microm). The number of lights (1, 2, 3, 4) and the angle of light (+1.5, 0, -3) were varied independently, creating 12 groups. Three images at each combination of settings were digitally captured and analyzed to obtain a histogram. The coefficient of resolution (Cr) was calculated to mathematically evaluate the grayscale histograms for intensities (0-255), where Cr = [¿IM - IN¿ x (N)] (IM, median pixel intensity; IN, measured pixel intensity; and N, number of pixels at given intensity). Mean Cr values demonstrated that the angle of light obliquity was not a factor in altering the resolution and contrast (p = .9). However, there was a significant increase in resolution, as measured by mean Cr values, as the number of lights was successively reduced from four lights to one light. Thus, the thicker specimen did show that increases in resolution were a significant function of the number of lights utilized.

A sheep model for thoracic aortic surgery in the presence of systemic coagulopathy.

Surgical repair of aneurysms, traumatic injuries, or congenital anomalies of the thoracic aorta are associated with high morbidity and mortality mainly as a result of excessive and uncontrollable hemorrhage from diffuse coagulopathy. We developed a model in sheep that simulates this coagulopathic state for experimentation with thoracic aorta surgery. This experimental animal model involves administering a 600-mg aspirin suppository once a day for the 2 days preceding surgery and a final dose on-call to surgery. Prior to cross-clamping the aorta, an intravenous (i.v.) bolus of heparin (400 IU/kg) was administered. Thirty minutes later, the i.v. heparin bolus was repeated. Pre- and intraoperative activated clotting time was 101 +/- 10 s and >1500 s (p < .0001); prothrombin time, 21 +/- 1 s and >100 s (p < .0001); and activated partial thromboplastin time, 20 +/- 1 s and >50 s (p < .0001), respectively. We utilized a partial cross-clamp-and-sew technique to anastomose a woven, gelatin-impregnated, 16-mm tube graft end-to-side to the descending thoracic aorta. Mean total blood loss was 1367 +/- 282 mL, which included mean blood loss from time of release of aortic cross-clamp to close (422 +/- 135 mL) and mean total blood output from chest tube drain (945 +/- 203 mL). The mean time to achieve hemostasis at suture lines after aortic cross-clamp release was 15.5 +/- 6.6 min. In conclusion, a sheep model with induced coagulation defects was successfully developed and reproducible for experimentation involving thoracic aortic surgery.

Vascularized bone marrow transplantation: A new surgical approach using isolated femoral bone/bone marrow.

BACKGROUND: Orthotopic composite tissue (limb) transplantation in rats is a unique model for vascularized bone marrow transplantation because bone marrow cells and bone marrow stroma are transplanted by microsurgical means, thus creating immediate bone marrow space and engraftment. However, it contains a skin component and other musculoskeletal tissues that complicate issues related to tolerance induction. MATERIALS AND METHODS: To study only aspects of vascularized bone marrow transplantation, we created a new isolated vascularized bone marrow transplant model in rats. The common iliac (or femoral) artery and vein were microsurgically anastomosed to the recipient abdominal aorta and inferior vena cava in an end-to-side fashion, respectively. Syngeneic male Lewis (RT1(1), n = 20) and allogeneic male BN (RT1(n), n = 10) donors were transplanted to female Lewis recipients. To establish rejection criteria, we examined histopathology and used the polymerase chain reaction (PCR) to assess microchimerism of donor male bone marrow cells in the peripheral blood of female recipients using rat Y chromosome (sex-determining region Y)-specific primers. RESULTS: All recipients were healthy and remained stable without major complications for up to 300 days posttransplant. Morphologically, syngeneic male Lewis bone marrow showed a near-normal appearance. Allogeneic male BN bone marrow was clearly rejected. Male bone marrow cells were detected by PCR in the peripheral blood of all syngeneic recipients, but not in allogeneic blood specimens. CONCLUSIONS: A new surgical approach to bone marrow transplantation was established. This consisted of the vascularized femoral bone/bone marrow transplant. Further analyses regarding the ability of vascularized femoral bone marrow transplants to induce systemic transplantation tolerance in adult rats will provide insights into not only various issues of immunology but also the potential clinical application of vascularized bone marrow transplantation.

Transmyocardial laser revascularization: current status.

Transmyocardial laser revascularization (TMLR) has been widely evaluated for treatment of the ischemic myocardium either in conjunction with coronary artery bypass grafting or as sole therapy. Clinically, it has shown significant improvement for angina symptoms, but the mechanism by which this modality works is unknown at this time. The original premise on which transmyocardial revascularization was established depended on its ability to essentially generate channels that would directly carry blood from the ventricle into the ischemic myocardium. This theory, however, has not been substantiated, so other mechanisms have been postulated. This article gives a historical perspective on the advent of transmyocardial revascularization and the many animal and human studies that have paved the way for its clinical use. Current controversies are examined, along with the new advances in laser technology and where the future of TMLR is headed.

Site-specific immunosuppression: mechanisms of cellular immunosuppression that are operative at local and systemic levels.

The cellular mechanisms by which topical cyclosporine A (tCsA) induces site-specific immunosuppression were investigated. Experiments were designed to elucidate how cyclosporine A (CsA) suppresses activated immunocytes in animals that are undergoing local alloactivation and concomitant tCsA immune suppression. Lewis rats received dual Lewis x Brown Norway rat skin allografts; the rats were treated with systemic CsA (sCsA) at 8 mg/kg/day for 10 days after grafting and then tCsA and vehicle thereafter. CsA added to mixed lymphocyte reactions 24 hours after culture initiation modeled the local effects of CsA on alloactivated immunocytes, and tCsA in conjunction with limited sCsA prolonged local skin allograft survival. CsA inhibited both antigen-specific and nonspecific activated alloresponses of immunocytes from animals that had received allografts and that underwent limited sCsA treatment only in a dose-dependent manner. When tCsA had been applied, immunocyte responses to a nonspecific antigen were extremely CsA-resistant as compared with those induced by antigen-specific suppression. However, this nonspecific alloresponse was fully suppressible with the use of elevated CsA doses (66 microg/mL); thus alloresponding immunocytes were significantly more sensitive to CsA if they were challenged with the donor antigen and preexposed to limited sCsA followed by tCsA in vivo.

An in vivo evaluation of a chondroitin sulfate solution to prevent postoperative intraperitoneal adhesion formation.

PURPOSE: The goal of this study was to determine the efficacy of a single intraperitoneal administration of a chondroitin sulfate solution in preventing postoperative adhesion formation. METHODS. Twenty-five Sprague-Dawley rats had a 1-cm(2) area of cecal serosa abraded. Controls (CON, n = 5) received no treatment, the chondroitin sulfate group (CS, n = 10) received chondroitin sulfate (0.013 g/kg) in 0.9% NaCl intraperitoneally (ip), and vehicle controls (VC, n = 10) received an equal volume of 0.9% NaCl solution ip before the abdomen was closed. All animals were sacrificed on postoperative day 10. The extent of adhesion was quantified according to Mazuji's adhesion grade (0 to 4: 0 = no adhesion and 4 = very dense adhesion) and quantitated after H&E, trichome, and immunohistochemical staining for fibrin and collagen type I and type III using digital image analysis. RESULTS: The mean Mazuji's adhesion grade in the CON was 4.0 +/- 0.0, in the VC 2.60 +/- 0.37, and in the CS 1.3 +/- 0.42 (P < 0.01 for CS vs CON and P < 0.05 for CS vs VC comparisons). The mean gray-scale intensity (0-255: 0 = dense amount and 255 = none) of adhesion density in the CON was 105. 5 +/- 5.5, in the VC 125 +/- 15.0, and in the CS 178.3 +/- 21.0 (P < 0.01 for CS vs CON and P < 0.05 for CS vs VC comparisons). The mean adjusted intensity stain indices (AISI) for fibrin and collagen type I in the CON were 59 +/- 17 and 53 +/- 19, in the VC 27 +/- 3 and 25 +/- 7, and in the CS 16 +/- 5 and 6 +/- 3, respectively (P < 0.05 between CS and CON comparisons). The AISI of collagen type III was not significant among all the groups (P > 0.1). CONCLUSIONS: The extent of early postoperative intra-abdominal adhesion formation as determined by gross assessment and from quantitation of fibrin and collagen type I deposition was significantly reduced by a single intraperitoneal administration of a chondroitin sulfate solution.

Past, present, and future research in the field of composite tissue allotransplantation.

In September 1998, a surgical team in Lyon, France, performed the first successful hand transplant. After this historic event, in January 1999, the University of Louisville performed the first hand transplant in the United States. These events sparked interest and debate concerning the justification of performing limb allotransplantation. The field of composite tissue allotransplantation (CTA) has made significant advances in the past two decades, yet advancement of the applications of CTA into the clinical arena had been fairly limited to this point. The most inherent controversy in CTA involves the fact that the clinical applications for the most part involve restoration of function and/or structural integrity. These procedures are done essentially for quality-of-life concerns, not life-saving issues. Present concern involves subjecting CTA recipients to a lifetime of postoperative immunosuppressive therapy. We cannot fully understand where we stand at present and in what future directions the field is heading unless we have an understanding of where we have been in composite tissue transplantation. This article reviews the historical aspects of CTA, discusses the present state of CTA, and speculates on potential future applications of CTA.

Composite tissue allografts in rats: IV. Graft-versus-host disease in recipients of vascularized bone marrow transplants.

This laboratory has used a composite tissue allograft model as a vehicle for studies on a new type of bone marrow transplant, the vascularized bone marrow transplant. The model consists of a rat hind limb transplant that incorporates integumentary musculoskeletal, and lymphopoietic tissues. These transplants, in comparison with conventional marrow transplants, have the advantage of providing a syngeneic microenvironment and immediate engraftment of both mature and progenitor hemopoietic cells at the time of transplantation. The characteristics of graft-versus-host disease were studied in this model. Lewis X Brown Norway F1 (LBN RT-1(1+n)) rats received hind limbs from Lewis (LEW RT-1(1)) donors (n = 19). Animals were observed daily for signs of graft-versus-host disease. Necropsies were performed. A minority of animals developed lethal disease (7 of 19 recipients) and demonstrated cachexia with concomitant histopathologic changes of the disease. Acute and chronic groups emerged with distinct clinical courses, which are similar to other models of this disease. Recipients of vascularized bone marrow transplants (limb transplants) showed clinical and histopathologic changes of the disease. The transplants may be used as a model of graft-versus-host disease in humans. Most interestingly, the transplant has a lower incidence of disease compared with other methods of bone marrow transplantation and represents an alternative to conventional bone marrow transplantation, which deserves further exploration. It may be possible to develop a new technique for bone marrow transplantation based on this surgical approach. It is proposed that the transfer of vascularized blocks of bone/marrow into prospective recipients as opposed to cellular bone marrow transplants may be preferable.

The effect of leukocyte infiltration on apoptosis in an in vitro thermal injury bioartificial living skin equivalent model.

An in vitro bioartificial skin construct (BSC) model was studied to see how inflammatory infiltration affects apoptosis in skin that has been thermally injured. The BSC was used as a target organ. Control BSCs without leukocytes (CON) were burned (BCON) by scalding with phosphate-buffered saline heated to 70 degrees C for 6 seconds, and they were then cooled with room temperature phosphate-buffered saline for 15 seconds. Human alloimmunocytes were added to CON to create rejection cultures (REJ) and to BCON to create burned rejection cultures (BREJ). Slides were stained with hematoxylin and eosin and anti-Lewis antibody. In situ labeling of apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Those sections that were immunostained for Lewis Y were analyzed for intensity stain index (ISI = [sigma P0 x I0]/ total tissue area in pixels). TUNEL was quantified with the following equation: no. of total apoptotic cells / total tissue area. Necrosis and blister formation in the epidermal layer were evident in BCON and BREJ. Pyknosis and nuclear fragmentation-indicators of apoptosis-were also present. Phagocytosis of keratinocytes by leukocytes was seen in REJ and BREJ. Immunostaining showed greater expression of Lewis Y antigen, as determined by ISI, in REJ as opposed to CON (58.2+/-2.3 vs. 36.4+/-2.3, respectively, P<.001), but no significant difference was found between BCON and BREJ (55.0+/-5.7 vs. 60.5+/-3.4, respectively) and REJ and BREJ (58.3+/-2.3 vs. 60.5+/-3.4, respectively). TUNEL staining indicated the presence of apoptosis as follows: REJ versus CON (0.0015+/-0.0002 vs. 0.0003+/-0.0001, respectively, P<.001); BREJ versus BCON (0.0031+/-0.0006 vs. 0.0018+/-0.0004, respectively, P<.05); REJ versus BREJ (0.0015+/-0.0002 vs. 0.0031+/-0.0006, respectively, P = .007). The presence of leukocytes and thermal injury induces apoptosis in BSC. The combination of these two variables results in increased apoptosis as determined by TUNEL. These findings suggest that a common pathway for skin injury may include inappropriate regulation of apoptosis exacerbated by a mechanism that includes inflammatory cellular infiltration.

Diamond-like carbon coating and plasma or glow discharge treatment of mechanical heart valves.

All mechanical heart valves (MHV) are thrombogenic. Application of surface modification technology to reduce the incidence of thrombus formation on MHV is a novel undertaking. This requires collaboration within the bioengineering and cardiothoracic surgery fields. From reviewing results of recent and past investigations, and our own preliminary study with diamond-like carbon coating (DLC) and plasma or glow discharge treatment (GDT) of MHV, we identify and discuss several potentially beneficial effects that may reduce the extent of valve-related thrombogenesis by surface modification. DLC and GDT may affect the surfaces of MHV in many ways, including cleaning of organic and inorganic debris, generating reactive and functional groups on the surface layers without affecting their bulk properties, and making the surfaces more adherent to endothelial cells and albumin and less adherent to platelets. These different effects of surface modification, separately or in combination, may transform the surfaces of MHV to be more thromboresistant in the vascular system.

Site-specific immunosuppression using a new formulation of topical cyclosporine A with polyethylene glycol-8 glyceryl caprylate/caprate.

PURPOSE: Dermal application of immunosuppressants can be an effective means of achieving site-specific immunosuppression (SITE) on skin allografts in burn wound management and in the treatment of various immune skin disorders. We have previously reported success with topical cyclosporine A (tCsA) in the treatment of skin allograft rejection in rats. Using a new tCsA formulation with a penetration enhancer (PE), polyethylene glycol-8 (PEG-8) glyceryl caprylate/caprate (Labrasol, Gattefossé, St. Priest, France), in a trinary drug delivery system, we hypothesized that we would induce SITE and significantly delay rejection of dual skin allografts in rats. METHODS: Dual rat skin allografts from Lewis x Brown-Norway (LBN) donors were grafted to Lewis (Lew) recipients. Experimental animals (EXP, n = 7) received a 10-day course of systemic cyclosporine (sCsA, 8 mg/kg/day) followed by topical application. One of the two allografts on each experimental animal received tCsA/PE application (5 mg/kg/day) until sacrifice (tCsA/PE-treated). The other allograft received vehicle only (vehicle-treated). Allogeneic controls (ALLO-CON, n = 9) received no sCsA or tCsA. First signs of rejection were determined based on the initial observation of erythema, hair loss, flakiness, and/or scabs. RESULTS: The mean time to rejection for ALLO-CON allografts was 6.3 +/- 0.7 days (t test, P = 0.0013); for vehicle-treated allografts, 12.3 +/- 3.8 days (paired t test, P = 0.0146); and for tCsA/PE-treated allografts, 25.6 +/- 5.4 days. The disparity of days to rejection between dual allografts in the ALLO-CON group was 0.0 +/- 0.0 day and that between the tCsA/PE- and vehicle-treated dual allografts was 13.3 +/- 3.9 days (t test, P = 0.0016). CONCLUSIONS: A new formulation of tCsA in a trinary drug delivery system is successful at delaying the onset of rejection in dual skin allografts in rats by SITE, and PEG-8 glyceryl caprylate/caprate may represent a potentially effective transdermal penetration enhancer.