Limb-saving surgery in osteosarcoma by vascularized fibular graft

Osteosarcoma is the most malignant bone tumor of the extremities that occurs in younger age groups; however, the prognosis of this disease has been improving. Limb-saving procedures have been successfully performed in more than half of the new cases of this disease. Because osteosarcoma occurs in young patients and their life expectancy is generally long, the rescued limbs must provide as full a service as possible for several decades. We have attempted the rescue of limbs in osteosarcoma patients, using the vascularized fibular graft (VFG). We performed a retrospective clinical and radiographic review of 12 patients with at least 3 years of follow up. The results were satisfactory in 11 out of the 12 patients from both the oncologic and functional point of view. There were no severe complications or donor site morbidity. Functional status, evaluated according to the system of Enneking et al. (Enneking WF, Dunham W, Gebhart MC, et al. A system for functional evaluation of reconstructive procedures after surgical treatment of tumors of the musculoskeletal system. Clin Orthop 1993;286:241–246), with some modifications exceeded 80%, except in one patient. The results of this study suggest that, because of the resulting durability of the reconstructed limb, VFG should be considered for limb rescue in young patients with osteosarcoma.     

Vascularized bone blocks from the toe phalanx to solve complex intercalated defects in the fingers

PURPOSE: Vascularized bone transplants resist infection and allow rapid healing but keeping small bony segments vascularized, as needed for a finger defect, is a challenge. The purpose of this article is to present a cohort of patients with traumatic intercalated compound bony defects in the fingers that were reconstructed by a vascularized toe phalanx (or part of a phalanx) in a single stage. METHODS: Eight patients were treated with an intercalary vascularized bone graft that included a part of the proximal phalanx (3 patients), most of the middle phalanx (4 patients), or a portion of each phalanx (1 patient) of a second toe (totaling 9 bone blocks). There was an associated soft-tissue defect in each patient, an infection in 6 patients, and cartilage loss in 4 patients. The toes were pedicled on the proper digital artery (6 patients) or a segment of the first dorsal metatarsal artery (2 patients). A mean length of 12 mm of vascularized bone was transferred. The associated skin island varied from a minimum of 2 x 1 cm to a maximum of 5 x 3 cm. Bleeding from all of the bone surfaces was evidenced once the clamps were released. The homolateral digital nerve and the contralateral neurovascular pedicle of the toe were kept in place. The toe defect was treated by soft-tissue arthroplasty or arthrodesis. No toe was amputated. RESULTS: Radiologic bony union was evident at 4 to 6 weeks, except in 1 patient with an acute infection whose distal union failed to unite at 6 weeks because the infection recurred. Finger length loss averaged 3 mm. All patients returned to their preoperative occupation. CONCLUSIONS: In this group of patients the toe phalanx reliably maintained its vascularization, allowing us to solve compound osteocutaneous defects in the fingers in a single stage. Donor site morbidity was minimal.     

Understanding of and attitudes to xenotransplantation: a survey among Italian university students

Xenotransplantation is currently at the experimental stages on animal models and many problems still have to be overcome in the biomedical, immunological and ethical fields. Moreover, people's attitudes to xenotransplantation vary: surveys among intensive-care staff have revealed negative opinions, while the general public and students seem to be more positive. Little is known about the influence of schooling and the choice of university faculty on attitudes to xenotransplantation. The aims of this study were: (i) to evaluate university students' attitudes to xenotransplantation; (ii) to investigate any socio-demographic, religious and educational determinants behind students' opinions on xenotransplantation. University undergraduates on five different courses were surveyed at Padua University. A 24-item questionnaire was distributed to students at the end of lectures and completed anonymously immediately after its distribution. No information was given to students beforehand. Statistical analysis: chi-squared, Pearson's test; P-values <0.05 were considered significant. A total of 585 of 602 (97.2%) students completed the questionnaire (132 males, 453 females, mean age 20.4, range 19 to 43 yr). They were on courses in Medicine (33.85%), Agriculture (5.98%), Veterinary Medicine (11.45%), Psychology (18.46%) and Educational Sciences (30.26%). As for their previous schooling, they came from classical or scientific high school (58.3%), technical college (14.7%), language college (6.3%), teacher training college (11.9%) or others (8.8%). Concerning their religious beliefs, 83% were Catholics, and 56.2% defined themselves as practising Catholics. Eighty-eight percentage of the students knew of the possibility of animal organs being transplanted into humans and 77.9% of them approved of this idea. When grouped according to gender and education, a higher proportion of students approving of xenotransplantation were male (P = 0.017) and had attended classical or scientific high school (P = 0.011). Disapproval for moral, ethical or religious reasons was higher among practising than among non-practising Catholics; the latter rejected xenotransplantation more for immunological and infectious reasons (P = 0.014). As for the type of university course, a higher proportion of students approving of xenotransplantation attended science courses (Veterinary Medicine, Agriculture and Medicine vs. Educational Sciences and Psychology) (P = 0.013). University students generally approved of xenotransplantation. Male gender and a high-school education were associated with a greater acceptance of xenotransplantation. Practising vs. non-practising Catholics reported significantly different reasons for any disapproval of xenotransplantation. The choice of a science rather than an arts faculty at university was more strongly associated with a positive opinion on xenotransplantation.     

Hospital personnel faced with organ xenotransplantation: an attitudinal survey in a hospital with a pre-clinical liver xenotransplantation program

BACKGROUND: In situations of extreme urgency when there is a need for vital organs, xenotransplantation could provide a bridge until the arrival of a human organ. However, it is important to find out the level of acceptance of this potential therapy among the health care workers who would be involved in its implementation. The objective of this study is to analyze attitude among personnel in a hospital with a pre-clinical xenotransplantation program toward xenotransplantation and to determine the variables that influence this attitude. MATERIALS AND METHODS: A random sample (n=1168) was taken and stratified according to job category and service. Attitude toward xenotransplantation was evaluated using a validated questionnaire. Contact was made with the head of each service who was given an explanation of the project. This person was made responsible for the distribution and collection of the survey in each service in randomly selected work shifts. Such a survey was completed anonymously and was self-administered. A random sample of 250 individuals from our regional community was used as a control group. RESULTS: The survey completion rate was 98% (n=1148). Most respondents are in favor (67%), 7% are against and 26% undecided. Such an attitude is more favorable in the control group (74% vs. 67%; P=0.0378). The following factors are positively related to such an attitude: (1) male sex (P<0.0005); (2) a younger age (P=0.013); (3) participation in prosocial voluntary activities (P=0.002); (4) knowing that the church has a positive attitude toward donation and transplantation (P<0.0005); (5) a partner's favorable attitude toward transplantation (P<0.0005); (6) a physician's job category (P<0.0005); (7) a resident physician's job contract situation (P=0.017); (8) a respondent's belief that he or she may need a transplant in the future (P<0.0005); and (9) a favorable attitude toward human donation, whether this be cadaveric or living (P<0.0005). In the multivariate analysis, the following persist as independent variables: (1) sex (odds ratio=1.6); (2) participation in prosocial voluntary activities (odds ratio=2.2); (3) a partner's unfavorable attitude toward transplantation (odds ratio=0.3); (4) a favorable attitude toward cadaveric donation (odds ratio=2); and (5) attitude toward living liver donation (odds ratio=3.8). CONCLUSIONS: Attitude toward xenotransplantation is not as favorable among hospital personnel as it is in the general public and this is determined by many factors. It will be necessary for research groups to periodically carry out awareness-raising activities about our findings in our own centers, to avoid the rejection that could be generated by a lack of awareness.     

A new autogenous graft choice in pelvic reconstructions: free vascularized rib (a case report)

Primary or secondary bone tumours are not uncommon in pelvic girdle. In some cases, after radical resection, there is a big bony defect where the prosthesis is not applicable; arthrodesis is the only choice for good functional results. In this instance, the major problem is to achieve the fusion. In this case report, we focused on easy harvesting and minimal time consumption with free vascularized rib graft to achieve the fusion between the resected segments. Two year follow up showed fusion with good functional result.     

Platelet aggregation in humans and nonhuman primates: relevance to xenotransplantation

Iwase H, Ekser B, Zhou H, Dons EM, Cooper DKC, Ezzelarab MB. Platelet aggregation in humans and nonhuman primates: relevance to xenotransplantation. Xenotransplantation 2012; 19: 233–243.. © 2012 John Wiley & Sons A/S. Abstract: Introduction: Platelet activation/aggregation plays a key role in the dysregulation of coagulation and the development of thrombotic microangiopathy in nonhuman primate recipients of pig xenografts. As a preliminary to the study of anti‐platelet therapy in vitro and in vivo, the present study aimed to compare platelet aggregation in whole blood from humans, baboons, and cynomolgus monkeys. Methods: Using “Chrono‐log” technology (two‐sample four‐channel Chrono‐log Whole Blood Aggregometer), we studied aggregation of platelets in healthy humans (n = 8), baboons (n = 5), and monkeys (n = 8). Whole blood (WB) samples were collected, and platelet aggregation was assessed using three different volumes of blood (1, 0.5, and 0.25 ml). Platelet activation was induced using collagen (at 3 and 5 μg/ml), ristocetin (at 0.5 and 1.0 mg/ml), adenosine diphosphate (ADP; at 10, 20, and 40 μm ), or thrombin (at 1 and 5 IU/ml). Inhibition of agonist‐induced platelet aggregation by heparin and low molecular weight heparin (LMWH) (at 1, 10, and 100 IU/ml) was evaluated. Results: Mean platelet counts were 222.1, 263.2, and 276.1 (×10³/μl) in humans, baboons, and monkeys, respectively. In all three species, platelet aggregation was induced by collagen, ristocetin, ADP, or thrombin in a dose‐dependent manner. A blood volume of 0.5 ml provided the most consistent results with all agonists in all three species. Dilution studies indicated that there was a significant positive correlation between platelet count and percent aggregation of platelets (P < 0.05). Collagen (3 and 5 μg/ml), ADP (10, 20, and 40 μm ), and thrombin (1 and 5 IU/ml) induced significantly greater platelet aggregation in humans than in baboons. ADP (20 and 40 μm ) and thrombin (1 and 5 IU/ml) induced significantly greater platelet aggregation in monkeys than in baboons. There was no species difference with ristocetin (0.5 or 1.0 mg/ml). In all species, thrombin (1 or 5 IU) induced greater platelet aggregation than any of the other reagents. Heparin at 1 IU/ml and LMWH at 10 IU/ml in all species almost completely abrogated thrombin‐induced platelet aggregation. Heparin at 100 IU/ml effectively inhibited platelet aggregation induced by collagen, but only partially inhibited aggregation induced by ADP or ristocetin. LMWH only partially inhibited aggregation induced by collagen, ristocetin, and ADP. Conclusions: The “Chrono‐log” technology proved to be a reliable method of evaluating platelet activation and aggregation in vitro in primates. Species differences may play a role in platelet aggregation, with the monkey being more comparable to the human than the baboon, although overall trends were similar. In all species, thrombin induced greater platelet aggregation than other agonists. Even a concentration of heparin of 1 IU/ml, which is probably the maximal concentration that is clinically‐applicable, prevented platelet aggregation induced by thrombin, but was less effective in preventing aggregation induced by collagen, ADP, or, particularly, ristocetin.     

Experimental study of vascularized bone grafts in rat: Effect of mechanical loading on bone dynamics

We studied the etiology of postoperative hypertrophy of vascularized bone grafts in a murine experimental model. Syngeneic grafting of revascularized ulna to rat tibia was performed with (group 1) or without (group 2) mechanical loading. The effect of simple overloading on intact bone was studied by segmental resection of the radius (group 3). Bone dynamics were examined by histomorphological measurements. Significant hypertrophy was observed in the early postoperative period in both groups 1 and group 2. After the initial phase, bone growth continued and extensive remodeling was observed in group 1, while marked bone resorption was observed in group 2. Adaptive remodeling was also observed in group 3 after surgery, but was slower than that in groups 1 and 2. Early hypertrophy of vascularized grafts did not correspond to mechanical loading. These results suggest that mechanical loading is the principal factor responsible for remodeling in vascularized bone grafts for their adaptation to a new environment.     

Approaching virus safety in xenotransplantation: a search for unrecognized herpesviruses in pigs

Abstract: The identification of porcine viruses so far unrecognized is required to minimize virus-related risks associated with xenotransplantation. We used a pan-herpes consensus polymerase chain reaction assay to search for unrecognized porcine species of the Herpesviridae. The assay targets conserved regions of the herpesvirus DNA polymerase (DPOL) gene, using primers that were modified to diminish the assay's recognition capacity for the highly prevalent porcine lymphotropic herpesviruses 1, 2 and 3 (PLHV-1, -2, -3), without substantially lowering the universal detection capacity of the assay. Analysis of 495 porcine blood and tissue samples from 294 animals, including 35 samples from 20 immunosuppressed pigs, resulted in the amplification of 128 herpesviral DPOL sequences. Sequence analysis attributed 127 of the amplimers to the known porcine herpesviruses (PLHV-1, -2, -3; porcine cytomegalovirus; pseudorabiesvirus). In none of the pig samples analyzed here, evidence was obtained for the presence of additional novel porcine herpesvirus species. Therefore we conclude that pigs bred for the purpose of xenotransplantation pose a negligible risk of transmitting presently unrecognized herpesviruses to organ recipients.     

Understanding of and attitude to xenotransplantation among Italian university students: impact of a 3-yr university course

BACKGROUND: Young people, and students in particular, generally have a positive attitude to xenotransplantation. We reported previously that university students attending their first year approved of the idea of transplanting animal organs into humans. The aim of this study was to evaluate the impact of a 3-yr course at various faculties on the university students' understanding of and attitude to xenotransplantation. METHODS: University students in their fourth year at five different faculties of Padua University (Italy), who had previously been surveyed in exactly the same way 3 yr earlier, were re-administered an anonymous 19-item questionnaire on their attitude to xenotransplantation. RESULTS: A total of 453 students completed the fourth year questionnaire (84 males, 369 females; mean age 24.6 yr, range 21-36 yr). Students were attending courses in Medicine (22.1%), Veterinary Science (16.5%), Agriculture (10.4%), Educational Sciences (22.1%) and Psychology (28.9%). In their fourth year, 85% of the students knew that animal organs could, at some stage, be transplanted into humans, 81.5% approved of this idea and 84% would accept an animal organ to save their life if necessary (these percentages were 88%, 78% and 76%, respectively 3 yr earlier). A significantly larger proportion of the students who approved of xenotransplantation were attending science courses rather than art courses, while no differences emerged as regards gender. Those who refused xenotransplantation justified their position mainly on ethical-moral (41.7%) and immunological (35.7%) grounds. CONCLUSION: As in their first year, so too in their fourth, University students were well informed about the feasibility of transplanting animal organs into human beings and those attending science courses were more likely to accept this idea than art students. Approval of xenotransplantation was much the same among fourth year males and females, whereas in the first year, male students had approved more than female students.     

Alpha-Gal detectors in xenotransplantation research: a word of caution

Naso F, Gandaglia A, Iop L, Spina M, Gerosa G. Alpha‐Gal detectors in xenotransplantation research: a word of caution. Xenotransplantation 2012; 19: 215–220. © 2012 John Wiley & Sons A/S. Abstract: Xenogeneic tissues are currently employed in clinical practice to create biological substitutes (bioprosthetic heart valves) and in the repair of various damaged tissues (pericardium, gastric‐mucosa, nerves, cartilage). Many studies have shown that xenogeneic tissues express superficial epitopes as alpha‐Gal, capable of triggering hyperacute and acute vascular rejection phenomena. Currently, no tissue treatment has proven able to completely mask or inactivate such epitopes. In fact, neither glutaraldehyde fixation nor decellularisation procedures ensure a definitive solution because of the persistence of reactive xenoantigen residues. The ability to ascertain alpha‐Gal epitope removal from a xenogeneic tissue is closely related to the possibility of its quantitative determination. In the past, detection of the alpha‐Gal epitope relied on the use of alpha‐Gal reactive isolectin molecules and was limited to isolated cells. Recently, the quantitative evaluation of this antigen has been carried out in whole connective tissue through the use of the monoclonal antibody M86. This article provides an overview of the implications of the alpha‐Gal epitope in the current clinical scenario and a definitive comparison between the reliability and specificity of isolectines vs. M86 in alpha‐Gal determination.     

Semi‐xenotransplantation: the regenerative medicine‐based approach to immunosuppression‐free transplantation and to meet the organ demand

Although xenografts have always held immeasurable potential as an inexhaustible source of donor organs, immunological barriers and physiological incompatibility have proved to be formidable obstacles to clinical utility. An exciting, new regenerative medicine‐based approach termed “semi‐xenotransplantation” (SX) seeks to overcome these obstacles by combining the availability and reproducibility of animal organs with the biocompatibility and functionality of human allografts. Compared to conventional xenotransplantation wherein the whole organ is animal‐derived, SX grafts are cleansed of their antigenic cellular compartment to produce whole‐organ extracellular matrix scaffolds that retain their innate structure and vascular channels. These scaffolds are then repopulated with recipient or donor human stem cells to generate biocompatible semi‐xenografts with the structure and function of native human organs. While numerous hurdles must be still overcome in order for SX to become a viable treatment option for end‐stage organ failure, the immense potential of SX for meeting the urgent needs for a new source of organs and immunosuppression‐free transplantation justifies the interest that the transplant community is committing to the field.     

Development of thymus autografts under the kidney capsule in the pig: A new "organ" for xenotransplantation

Abstract: Ten piglets, 7 to 16 weeks old, were partially thymectomised and 1 to 4 cm³ of minced thymic fragments autografted under the renal capsule. They were sacrificed, respectively, after 2, 4, 6, 8, 12, and 20 weeks. After 2 weeks, irregular whitish zones are present under the renal capsule. They were composed principally of two cell types: the first type was characterized by small round basophilic nuclei and little cytoplasm typical of lymphocytes; the second cell type had larger ovoid nuclei and a large vacuolised cytoplasm. Each cell type could be found in separate lobules or mixed in variable proportion in the same structure. The thymic autografts grew to form a layer up to 4 mm thick after 20 weeks. In the meantime, at the beginning of 4th week, the lobular structure became well organized with the cell type presenting large nuclei and cytoplasm being restricted to the center of the lobules while lymphocytes composed a peripheral layer. Hassal corpuscles (HC) appeared in the center of the lobules. Immunohistochemical labeling with anti-cytokeratin mono- and poly- clonal Ab and with anti-neurophysin polyclonal Ab displayed all the characteristics of normal functional thymic microenvironment. It is proposed that this novel experimental preparation ending up as a neo-organ (thymo-kidney) be used for xenotransplantation in an attempt to produce specific xenotolerance.     

Recipient‐derived angiogenesis with short term immunosuppression increases bone remodeling in bone vascularized composite allotransplantation: A pilot study in a swine tibial defect model

Current vascularized composite allotransplantation (VCA) transplantation protocols rely upon life‐long immune modulation to maintain tissue perfusion. Alternatively, bone‐only VCA viability may be maintained in small animal models using surgical angiogenesis from implanted autogenous vessels to develop a neoangiogenic bone circulation that will not be rejected. This study tests the method's efficacy in a large animal model as a bridge to clinical practice, quantifying the remodeling and mechanical properties of porcine tibial VCAs. A segmental tibial defect was reconstructed in Yucatan miniature swine by transplantation of a matched tibia segment from an immunologically mismatched donor. Microsurgical repair of nutrient vessels was performed in all pigs, with simultaneous intramedullary placement of an autogenous arteriovenous (AV) bundle in Group 2. Group 1 served as a no‐angiogenesis control. All received 2 weeks of immunosuppression. After 16 weeks, micro‐CT and histomorphometric analyses were used to evaluate healing and remodeling. Axial compression and nanoindentation studies evaluated bone mechanical properties. Micro‐CT analysis demonstrated significantly more new bone formation and bone remodeling at the distal allotransplant/recipient junction and on the endosteal surfaces of Group 2 tibias (p = 0.03). Elastic modulus and hardness were not adversely affected by angiogenesis. The combination of 2 weeks of immunosuppression and autogenous AV‐bundle implantation within a microsurgically transplanted tibial allotransplant permitted long‐term allotransplant survival over the study period of 16 weeks in this large animal model. Angiogenesis increased bone formation and remodeling without adverse mechanical effects. The method may allow future composite‐tissue allotransplantation of bone without the risks associated with long‐term immunosuppression. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1242–1249, 2017.

     

Cryopreserved human skin allografts promote angiogenesis and dermal regeneration in a murine model

Cryopreserved human skin allografts (CHSAs) are used for the coverage of major burns when donor sites for autografts are insufficiently available and have clinically shown beneficial effects on chronic non‐healing wounds. However, the biologic mechanisms behind the regenerative properties of CHSA remain elusive. Furthermore, the impact of cryopreservation on the immunogenicity of CHSA has not been thoroughly investigated and raised concerns with regard to their clinical application. To investigate the importance and fate of living cells, we compared cryopreserved CHSA with human acellular dermal matrix (ADM) grafts in which living cells had been removed by chemical processing. Both grafts were subcutaneously implanted into C57BL/6 mice and explanted after 1, 3, 7, and 28 days (n = 5 per group). A sham surgery where no graft was implanted served as a control. Transmission electron microscopy (TEM) and flow cytometry were used to characterise the ultrastructure and cells within CHSA before implantation. Immunofluorescent staining of tissue sections was used to determine the immune reaction against the implanted grafts, the rate of apoptotic cells, and vascularisation as well as collagen content of the overlaying murine dermis. Digital quantification of collagen fibre alignment on tissue sections was used to quantify the degree of fibrosis within the murine dermis. A substantial population of live human cells with intact organelles was identified in CHSA prior to implantation. Subcutaneous pockets with implanted xenografts or ADMs healed without clinically apparent rejection and with a similar cellular immune response. CHSA implantation largely preserved the cellularity of the overlying murine dermis, whereas ADM was associated with a significantly higher rate of cellular apoptosis, identified by cleaved caspase‐3 staining, and a stronger dendritic cell infiltration of the murine dermis. CHSA was found to induce a local angiogenic response, leading to significantly more vascularisation of the murine dermis compared with ADM and sham surgery on day 7. By day 28, aggregate collagen‐1 content within the murine dermis was greater following CHSA implantation compared with ADM. Collagen fibre alignment of the murine dermis, correlating with the degree of fibrosis, was significantly greater in the ADM group, whereas CHSA maintained the characteristic basket weave pattern of the native murine dermis. Our data indicate that CHSAs promote angiogenesis and collagen‐1 production without eliciting a significant fibrotic response in a xenograft model. These findings may provide insight into the beneficial effects clinically observed after treatment of chronic wounds and burns with CHSA.