A double chamber rotating bioreactor for enhanced tubular tissue generation from human mesenchymal stem cells: a promising tool for vascular tissue regeneration.

Cardiovascular diseases represent a major global health burden, with high rates of mortality and morbidity. Autologous grafts are commonly used to replace damaged or failing blood vessels; however, such approaches are hampered by the scarcity of suitable graft tissue, donor site morbidity and poor long-term stability. Tissue engineering has been investigated as a means by which exogenous vessel grafts can be produced, with varying levels of success to date, a result of mismatched mechanical properties of these vessel substitutes and inadequate ex vivo vessel tissue genesis. In this work, we describe the development of a novel multifunctional dual-phase (air/aqueous) bioreactor, designed to both rotate and perfuse small-diameter tubular scaffolds and encourage enhanced tissue genesis throughout such scaffolds. Within this novel dynamic culture system, an elastomeric nanofibrous, microporous composite tubular scaffold, composed of poly(caprolactone) and acrylated poly(lactide-co-trimethylene-carbonate) and with mechanical properties approaching those of native vessels, was seeded with human mesenchymal stem cells (hMSCs) and cultured for up to 14 days in inductive (smooth muscle) media. This scaffold/bioreactor combination provided a dynamic culture environment that enhanced (compared with static controls) scaffold colonization, cell growth, extracellular matrix deposition and in situ differentiation of the hMSCs into mature smooth muscle cells, representing a concrete step towards our goal of creating a mature ex vivo vascular tissue for implantation. Copyright © 2016 John Wiley & Sons, Ltd.

Autosomal recessive osteopetrosis: report of 41 novel mutations in the TCIRG1 gene and diagnostic implications.

UNLABELLED: Here we report 41 novel mutations in the TCIRG1 gene that is responsible for the disease in more than 50% of ARO patients. The characterisation of mutations in this gene might be useful in the process of drug design for osteoporosis treatment. INTRODUCTION: Autosomal recessive osteopetrosis (ARO) is a genetically heterogeneous disorder due to reduced bone resorption by osteoclasts. In this process, a crucial role is played by the proton pump V-ATPase. Biallelic mutations in the TCIRG1 gene, encoding for the a3 subunit of this pump, are responsible for more than one half of ARO patients. METHODS: Patients with a clinical diagnosis of ARO have been collected for 7 years and mutation analysis of the TCIRG1 gene was performed using direct DNA sequencing of PCR-amplified exons according to both a standard protocol and a modified one. RESULTS: We report here 41 novel mutations identified in 67 unpublished patients, all with biallelic mutations. In particular, we describe two novel large genomic deletions and two splice site mutations in the 5' UTR of the TCIRG1 gene, in patients previously classified as mono-allelic. CONCLUSIONS: Our data highlights the importance of two large genomic deletions and mutations in the 5' UTR with respect to patient management and, more critically, to prenatal diagnosis. With the present work, we strongly contribute to the molecular dissection of TCIRG1-deficient ARO and identify several protein residues which are fundamental for proton pump function and could thus be the target of future drugs designed to inhibit osteoclast resorptive activity.

Bioelectrical Impedance Vector Analysis and Mini Nutritional Assessment in elderly nursing home residents.

OBJECTIVE: The aim of this study was to establish the presence of malnutrition in elderly institutionalized people through Mini Nutritional Assessment (MNA) scores and to observe the changes of nutritional status through the Bioelectrical Impedance Vector Analysis (BIVA). DESIGN: Observational study. SETTING: Nursing home located in the territory of Florence (Italy). PARTICIPANTS: 463 elderly individuals from twelve nursing homes, all aged 65 yrs or over. MEASUREMENTS: We carried out the MNA and measured the bioelectrical parameters of resistance (R) and reactance (Xc), with standard, tetrapolar analysis at 50 kHz frequency; these bioelectrical parameters were interpreted according to BIVA analysis. RESULTS: According to the MNA score, 23.1% of the women and 20.4% of the males are malnourished (MNA < 17), 60.0% and 52.8%, respectively, are at risk for malnutrition (17 ≤ MNA ≤ 23.5). Malnourished, at-risk and well-fed subjects differ significantly (p < 0.05) in their mean scores obtained in each of the MNA areas. Fifteen of eighteen MNA items show a significant correlation to the total MNA score (p < 0.001). The items not significantly related to the MNA global score were those concerning protein intake, independence and number of medicinal drugs taken. In both genders, the decrease of the MNA score increases the proportion of subjects placed outside the 95% confidence ellipse on the BIVA tolerance graph. CONCLUSION: A routine assessment of nutritional status through BIVA analysis combined with more "traditional" tools like the MNA could be useful for recognizing malnutrition and the risk of malnutrition in the elderly as these instruments provide complete information and are easy to use even with heavily impaired subjects.

Skeletal myogenesis on highly orientated microfibrous polyesterurethane scaffolds.

Skeletal myogenesis is a complex process, which is known to be intimately depending on an optimal outside-in substrate-cell signaling. Current attempts to reproduce skeletal muscle tissue in vitro using traditional scaffolds mainly suffer from poor directionality of the myofibers, resulting in an ineffective vectorial power generation. In this study, we aimed at investigating skeletal myogenesis on novel biodegradable microfibrous scaffolds made of DegraPol, a block polyesterurethane previously demonstrated to be suitable for this application. DegraPol was processed by electrospinning in the form of highly orientated ("O") and nonorientated ("N/O") microfibrous meshes and by solvent-casting in the form of nonporous films ("F"). The effect of the fiber orientation at the scaffold surface was evaluated by investigating C2C12 and L6 proliferation (via SEM analysis and alamarBlue test) and differentiation (via RT-PCR analysis and MHC immunostaining). We demonstrated that highly orientated elastomeric microfibrous DegraPol scaffolds enable skeletal myogenesis in vitro by aiding in (a) myoblast adhesion, (b) myotube alignment, and (c) noncoplanar arrangement of cells, by providing the necessary directional cues along with architectural and mechanical support.

[Body composition, nutritional habits and functional abilities in a sample of eight year old children]. / Composizione corporea, abitudini alimentari e abilita funzionali in un campione di bambini di 8 anni.

Aim of this study is the evaluation of body composition (proxy of nutritional status) and its correlation with functional abilities in a sample of 8-year-old children living in a florentine municipality. We have collected, for each subject, anthropometric variables (weight, height) and carried out bioelectrical impedance test and a group of physical tests to measure strength and physical ability. The results of vectorial interpretation of BIA, together with the analysis of the so called fitness tests, show a comprehensive good body composition and a very low prevalence of obesity for both genders. This fact is confirmed also by data about living and nutrition habits: scarce use of PC and TV (less than two hours a day) and, on the other hand, enough time spent to play in open spaces and to make sport.

[Fatal home accidents in Tuscany in 2001-2002]. / Incidenti mortali domestici in Toscana negli anni 2001-2002.

In the last years in Italy deaths due to accidents were more than 26.000/year. One of the most frequent places in which they occurred was at home, with more than 8.000 deaths/year. Due to the lack of studies related to this field, in Tuscany a specific study of fatal home accidents has been done. The cases studied, occurred in Tuscany in 2001-2002, have been extracted from the electronic database of the Regional Mortality Registry of Tuscany. Information about the modality of the accidents was collected by administering a questionnaire to the closest available relative of the victim. A total of 284 cases were studied, the majority of whom were female, over 75 years of age, with low educational level, pensioners, often with more than three pathologies. The accidents happened mostly in the bedroom or on the stairs due to accidental falls. The dynamics of fatal accidents showed great differences if compared to the nonfatal accidents. This suggests the need of different approaches for fatal injury surveillance and for the implementing of preventive strategies.

A new electro-mechanical bioreactor for soft tissue engineering.

By enabling the maintenance of controlled chemical and physical environmental conditions, bioreactors proved that electro-mechanical stimulation improves tissue development in vitro, especially in the case of tissues which are subjected to stimuli during embryogenesis and growth (i.e. skeletal and cardiac muscle tissue). However, most of the bioreactors developed in the last 20 yrs, designed to suit specific applications, lack versatility. With the aim to provide researchers with a yielding, versatile tool, we designed and realized in this study an electro-mechanical stimulator capable of dynamically culturing four biological constructs, delivering assignable stretching and electrical stimulation patterns. The device has been conceived to be easy to handle and customizable for different applications, while ensuring sterility along with stimuli delivery. The gripping equipment, modular and adaptable to scaffolds of different consistencies, is provided with dedicated tools for supporting sample insertion into the culture chamber performed under a laminar flow hood. As to performance, a wide range of electro-mechanical stimulation patterns and their relative occurrence can be accomplished, permitting the adjustment of the dynamic culture parameters both to the specific cell species and to the developmental phase of the cultured cells.

Mechanical properties of breast periprosthetic capsules and the correlation to capsule contracture.

Contracture of the fibrous connective capsules that form around breast implants is the most frequent complication for silicone mammary prostheses. Capsule contraction is thought to be associated with the alteration in the mechanical properties of the tissue, but to date such a correlation has not been quantified. This study aimed to develop an experimental protocol to investigate the mechanical properties of the periprosthetic tissue and their dependence on the severity of capsular ontracture. Capsule samples were harvested from donors undergoing surgical correction or re-implantation of breast prostheses; strip spec-imens were cut along both the meridian and circumferential directions of the capsular dome. The experimental protocol consisted of mechanical preconditioning, stress relaxation tests and tensile tests to rupture. The capsular tissue exhibited non-linear stress-strain relationships and stress relaxation behavior; nevertheless, both the elastic and the relaxation properties did not depend on the loading direction. In spite of the small number of samples included, to date, in the experimentation, some pre-liminary conclusions can be drawn: the stiffness and the strength of the tissue increases with the contracture severity; the re-laxation seems unaffected by the contracture degree. Some assumptions concerning the modifications in periprosthetic tissue composition following contracture are also drawn.

Endothelial cell adhesion force estimation at the nanoscale.

Endothelial cell adhesion to a synthetic surface includes a definite set of molecular interactions. Cell adhesion is managed by fibronectin and vitronectin in extracellular matrix (ECM) that binds the receptor site of the trans-membrane protein dimers, the integrins. These proteins contain one of the binding sites (I-like domain receptor) for the Arg-Gly-Asp (RGD) peptides that are the established adhesion receptor sites in the ECM. A molecular approach can quantify the adhesion strength by ligand-receptor force computation. The molecular interaction energy between a polyethylene (PE) surface covalently grafted with the adhesion sites (RGD-containing) and trans-membrane integrin receptor (I-domain) was evaluated through a molecular model of a single ligand-receptor complex. The aims of this work were: (i) the generation of the receptor molecular model: the I-like domain; (ii) the evaluation of the greatest binding chemical affinity between the I-like domain and three RGD-containing peptides; (iii) the development of the molecular model of crystalline lamellae of PE; and (iv) the evaluation of the interac-tion energies and the interaction force between the I-like domain and the grafted biomaterial. The calculation of the interaction energies can provide an estimation of the adhesion force of the ligand-receptor complex and, finally, of the endothelial cell adhesion force. The calculated cell adhesion force is in agreement with experimental data.(Journal of Applied Biomaterials and Biomechanics 2005; 3: 42-9).

Possible role of decorin glycosaminoglycans in fibril to fibril force transfer in relative mature tendons--a computational study from molecular to microstructural level.

Experimental studies on immature tendons have shown that the collagen fibril net is discontinuous. Manifold evidences, despite not being conclusive, indicate that mature tissue is discontinuous as well. According to composite theory, there is no requirement that the fibrils should extend from one end of the tissue to the other; indeed, an interfibrillar matrix with a low elastic modulus would be sufficient to guarantee the mechanical properties of the tendon. Possible mechanisms for the stress-transfer involve the interfibrillar proteoglycans and can be related to the matrix shear stress and to electrostatic non-covalent forces. Recent studies have shown that the glycosaminoglycans (GAGs) bound to decorin act like bridges between contiguous fibrils connecting adjacent fibril every 64-68 nm; this architecture would suggest their possible role in providing the mechanical integrity of the tendon structure. The present paper investigates the ability of decorin GAGs to transfer forces between adjacent fibrils. In order to test this hypothesis the stiffness of chondroitin-6-sulphate, a typical GAG associated to decorin, has been evaluated through the molecular mechanics approach. The obtained GAG stiffness is piecewise linear with an initial plateau at low strains (<800%) and a high stiffness region (3.1 x 10(-11)N/nm) afterwards. By introducing the calculated GAG stiffness in a multi-fibril model, miming the relative mature tendon architecture, the stress-strain behaviour of the collagen fibre was determined. The fibre incremental elastic modulus obtained ranges between 100 and 475 MPa for strains between 2% and 6%. The elastic modulus value depends directly on the fibril length, diameter and inversely on the interfibrillar distance. In particular, according to the obtained results, the length of the fibril is likely to play the major role in determining stiffness in mature tendons.

An approach to computer automation of the extracorporeal circulation.

In order to move towards extracorporeal circulation (ECC) automation, a virtual simulation of the process was designed. The ECC model is composed of a virtual patient linked to a virtual ECC circuit. A user interface panel allows to set control parameters for the simulation and to visualize results. It is possible to switch between manual and automatic control. Meaningful hemodynamic and hematochemical variables are continuously shown along with a score (from 0 to 10). The virtual model can play a crucial role in educating and training the personnel devoted to the managing of the heart-lung machine.

Multiple functions of Dlx genes.

Dlx genes comprise a highly conserved family of homeobox genes homologous to the distal-less (Dll) gene of Drosophila. They are thought to act as transcription factors. All Dlx genes are expressed in spatially and temporally restricted patterns in craniofacial primordia, basal telencephalon and diencephalon, and in distal regions of extending appendages, including the limb and the genital bud. Most of them are expressed during morphogenesis of sensory organs and during migration of neural crest cells and interneurons. In addition, Dlx5 and Dlx6 are expressed in differentiating osteoblasts. Gene targeting of Dlx1, Dlx2, Dlx3 and Dlx5 in the mouse germ-line has revealed functions in craniofacial patterning, sensory organ morphogenesis, osteogenesis and placental formation. However, no effect on limb development has yet been revealed from gene inactivation studies. A role for these genes in limb development is however suggested by the linkage of the Split Foot/Hand Malformation human syndrome to a region containing DLX5 and DLX6. As for most transcription factors, these genes seem to have multiple functions at different stages of development or in different tissues and cell types.

Rapid crossing of the pulmonary endothelial barrier by polyethylenimine/DNA complexes.

Intravenous administration could become a delivery route of choice for prophylactic and curative gene therapies on condition that genes cross the capillary barrier and reach target tissues without being degraded. We investigated the kinetics and process of transgene delivery through mouse lung capillaries following DNA complexation with linear polyethylenimine (L-PEI) and intravenous injection. Using digoxin-labeled DNA we followed the cellular localization of DNA at different times after injection and correlated these findings with cell markers and transgene expression. At 2 h after injection some DNA was still localized on the interior of the capillary lumen, but other complexes had already crossed the barrier and resulted in gene expression. At 24 h after injection most labeled DNA was localised in pulmonary cells, as was transgene expression. Only rarely was transgene expression found in endothelial cells, suggesting that the complexes cross the capillary barrier rapidly. Levels of caspase-1-like activity did not increase following transfection implying that L-PEI/DNA complexes are transported across cellular barriers by a non-damaging, physiological process, without causing inflammation. The high levels of expression of different transgenes in pneumocytes indicates that transport of L-PEI/DNA complexes through the endothelial barrier does not affect their transfection capacity. These findings open up new possibilities for gene delivery and its application to the lung.

Hyperplasia and impaired involution in the mammary gland of transgenic mice expressing human FGF4.

Fgf4, a member of the fibroblast growth factor family, is frequently amplified in a variety of human cancers, however, its expression in neoplastic tissues is rarely detectable. This makes uncertain its involvement in tumour aetiology, although several in-vitro studies link Fgf4 overexpression to malignant transformation and metastatization of culture cells. We generated a transgenic mouse model in which the whey acidic protein (WAP) promoter directs expression of human Fgf4 to mammary tissues during late pregnancy and throughout lactation, with the purpose of studying the involvement of this growth factor in mammary tumorigenesis. Expression of the transgene was specifically detected in lobular-alveolar cells of lactating mammary glands that, by histological analysis, displayed hyperplastic areas and a disorganized structure. This was accompanied by an increased number of red blood cells and expression, in alveolar epithelial cells, of the vascular endothelial growth factor, which is absent in wild type controls. The most striking effect caused by FGF4 overexpression was on the remodelling of mammary tissue at the end of lactation. Indeed, transgenic animals showed a delayed involution of the gland due to a dramatic reduction in the overall number of apoptotic cells, which are normally present in the organ after weaning. Nevertheless, none of the animals examined developed neoplastic lesions of the mammary gland even after several pregnancies and at old age. Our work represents the first in-vivo demonstration of the anti-apoptotic and angiogenic properties of FGF4.

In vivo study of polyurethane-coated Gianturco-Rosch biliary Z-stents.

PURPOSE: Prototypes of Gianturco-Rosch Z-stents coated with polycarbonate urethane (PCU) were placed in the biliary tree of pigs, in order to test their biomechanical behavior, stability, and biocompatibility. METHODS: The stents were surgically implanted in the common bile duct of three pairs of pigs, which were killed after 1, 3, and 6 months respectively. Explanted livers from pigs of the same race, age, and size were used to provide comparative data. The bile ducts were radiologically and histopathologically examined; the stents were processed and examined by scanning electron microscopy. RESULTS: No complications occurred and the animals showed a normal weight gain. The main bile duct appeared radiologically and macroscopically dilated, but the stents proved to be in place. Histologically, the bile duct epithelium was destroyed, but neither hyperplastic nor inflammatory fibrotic reactions of the wall were evident. Both the metallic structure and the polymeric coating of the stents were intact. A layer of organic material with a maximum thickness of approximately 3 micron was evident on the inner surface of the stents. CONCLUSION: The present in vivo study demonstrates the biocompatibility, efficacy, and stability of PCU-coated Gianturco-Rosch stents in the biliary environment.

Effects of cooling micrografts in hair transplantation surgery.

BACKGROUND: When isolating hair grafts in hair transplantation procedures, it is generally recommended to preserve the grafts at a low temperature (1 degrees C- 4 degrees C) in order to enhance the survival rate of the grafted hairs. This study was carried out to test the real benefits provided by cold-storing hair grafts. OBJECTIVE: The aim of this study was to evaluate, in an in vitro model, the effects of cooling micrografts during hair transplantation procedures to experimentally assess the soundness of this approach to enhance the survival and growth rates of hair micrografts. METHODS: A total of 240 anagen hair follicles were obtained from 10 healthy male patients. Follicles were thus randomly assigned to one of the following groups: group A (control; n = 120 follicles), or group B (experimental; n = 120 follicles). Follicles from group A were preserved for 5 hours at room temperature (26 degrees C), and follicles from group B were preserved for 5 hours at 1 degrees C. Immediately after that 5-hour period, follicles from both groups were then cultured for 10 days. The length of each follicle was measured immediately following the 5-hour test period and at the end of the 10-day culture period. RESULTS: No statistically significant differences were found between the survival and growth rates of follicles from the control (survival rate = 87%, mean 10-day growth rate = 2.68 mm) and experimental (survival rate = 88%, mean 10-day growth rate = 2.54 mm) groups. CONCLUSION: Although, at present, it is generally assumed that lowering the metabolism of grafts by reducing their temperature may be of some utility for enhancing their survival rate, our data indicate of that there are no effects when performing hair transplantation surgery.

Craniofacial, vestibular and bone defects in mice lacking the Distal-less-related gene Dlx5.

The Dlx5 gene encodes a Distal-less-related DNA-binding homeobox protein first expressed during early embryonic development in anterior regions of the mouse embryo. In later developmental stages, it appears in the branchial arches, the otic and olfactory placodes and their derivatives, in restricted brain regions, in all extending appendages and in all developing bones. We have created a null allele of the mouse Dlx5 gene by replacing exons I and II with the E. coli lacZ gene. Heterozygous mice appear normal. Beta-galactosidase activity in Dlx5+/- embryos and newborn animals reproduces the known pattern of expression of the gene. Homozygous mutants die shortly after birth with a swollen abdomen. They present a complex phenotype characterised by craniofacial abnormalities affecting derivatives of the first four branchial arches, severe malformations of the vestibular organ, a delayed ossification of the roof of the skull and abnormal osteogenesis. No obvious defect was observed in the patterning of limbs and other appendages. The defects observed in Dlx5-/- mutant animals suggest multiple and independent roles of this gene in the patterning of the branchial arches, in the morphogenesis of the vestibular organ and in osteoblast differentiation.

Lumbar dura mater biomechanics: experimental characterization and scanning electron microscopy observations.

UNLABELLED: There is no consensus about the anatomical structure of human dura mater. In particular, the orientation of collagen fibers, which are responsible for biomechanical behavior, is still controversial. The aim of this work was to evaluate the mechanical properties and the microstructure of the lumbar dura mater. We performed experimental mechanical characterization in longitudinal and circumferential directions and a scanning electron microscopy observation of the tissue. Specimens of human dura mater were removed from the dorsal-lumbar region (T12-L4/L5) of six subjects at autopsy; specimens of bovine dorsal-lumbar dura mater were obtained from two animals at slaughter. Human and bovine tissues both exhibited stronger tensile strength and stiffness in the longitudinal than in the circumferential direction. Scanning electron microscopy observations of dura mater showed that the collagen fibers are mainly oriented in a longitudinal direction, which accounts for its stronger tensile strength in this direction. We conclude that dura mater has a different mechanical response in the two directions investigated because the fiber orientation is predominantly longitudinal. IMPLICATIONS: In this experimental work, we studied the structural and functional relationship of human lumbar dura mater. We performed mechanical tests and microscopic observations on dura mater samples. The results show that the dura mater is mainly composed of longitudinally oriented collagen fibers, which account for higher tissue resistance in this direction.

Evaluation of temperature distribution during hyperthermic treatment in biliary tumors: a computational approach.

A computational approach is adopted to predict the temperature distribution in the biliary tissue during hyperthermic treatments in biliary tumors. Two different models are developed: an axisymmetric model and a three-dimensional model. In the first model the Pennes bioheat transfer equation is applied. It is aimed at simulating the thermoregulatory effect of the capillary bed and it can also give a pressure criterion to determine whether the blood perfusion term should be included in the mathematical model. The second model is aimed at simulating the convective effect of the large hepatic vessels: A constant Nusselt number is assumed on the sides of the vessels. The simulations of the three-dimensional model have been carried out with and without capillary perfusion in the tissue, i.e., respectively in the worst case and in the best case that may occur during heating. The results show that it is possible to obtain therapeutic temperature values in the tissue for time intervals considered acceptable by physicians. Moreover, the model is able to give more precise information about the volumes of tumoral tissue heated above therapeutic temperatures with the hyperthermic technique considered.

Fresh and cryopreserved arterial homografts in the treatment of prosthetic graft infections: experience of the Italian Collaborative Vascular Homograft Group.

Following the experience of cardiac surgeons with homografts in the treatment of infective aortic valve endocarditis, cardiovascular surgeons have investigated in situ revascularization by means of homografts in the management of vascular prosthetic graft infections. Preliminary results are encouraging, but their late fate in long-term follow-up and the influence of preservation techniques are still under investigation. This article reports the experience of the Italian Collaborative Vascular Homograft Group, with the use of fresh and cryopreserved arterial homografts for the treatment of prosthetic graft infections. Between March 1994 and December 1996, 44 patients with prosthetic graft infection were treated with homografts (13 preserved at 4 degrees C, 31 cryopreserved). The mean age of the patients was 65 years. Emergency surgical procedures were performed in eight patients (18%). Sepsis was diagnosed in 11 patients, aortoenteric fistula in 13, and false aneurysms in 10. Staphylococcus was the main cause of infection. The types of vascular reconstruction with homograft were: 32 aortobifemoral, 3 aortoaortic, 2 iliofemoral, 4 peripheral, and 3 axillobifemoral. Human lymphocyte antigen (HLA) and antibody (ABO) blood group system compatibility between donors and recipients was not respected. The mean duration of follow-up was 15 months (range 1-33). Clinical and duplex scanning evaluations were routinely performed. Computed tomography (CT) or magnetic resonance (MR) scanning or arteriography were performed on the basis of duplex scanning results. There were six deaths during the early postoperative period (30 days) with a mortality rate of 13.6%. During the follow-up there were five late deaths with a mortality rate of 11.4%. Eight patients had graft occlusion. Three cases were successfully treated with thrombectomy. Two cases were successfully treated with femoropopliteal bypass with autologous vein. In three cases leg amputation was necessary. The results of fresh and cryopreserved homograft were compared. No significative differences of early postoperative mortality, late mortality, homograft related mortality, and graft occlusion were observed. We have evaluated the actuarial survival of the patients and the actuarial patency of the homografts on the aortoiliac reconstructions. Twelve months after the surgery the actuarial survival of the patients was 73% and the actuarial patency of the homografts was 56%. In our preliminary experience, we have not observed any significant difference in terms of clinical outcome by using fresh rather than cryopreserved homografts. In the near future it will be our policy to employ only cryopreserved homografts. Moreover, we will extend vessel harvesting to nonheart-beating donors, thus maximizing retrieval. The aforementioned solutions will supply the best graft availability to obtain dimensional and ABO compatibility between donors and recipients.