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The main purpose of tissue engineering is to fabricate and exploit engineered constructs suitable for the effective replacement of damaged tissues and organs, and able to perfectly integrate with the host’s organism without eliciting any adverse reaction. Ideally, autologous materials represent the best option, but they are often limited due to the low availability of compatible healthy tissues. So far, one therapeutic approach relies on the exploitation of synthetic materials: they exhibit good features in terms of impermeability, deformability and flexibility, but present chronic risks of infections and inflammations. Alternatively, biological materials, including naturally derived ones and acellular tissue matrices of human or animal origin, can be used to induce cells growth and differentiation, which are needed for tissue regeneration: however, this kind of materials lacks satisfactory mechanical resistance and reproducibility, affecting their clinical application. In order to overcome the above-mentioned limitations, hybrid materials, which can be obtained by coupling synthetic polymers and biological materials, have been investigated with the aim to improve biological compatibility and mechanical features. Currently, the interest in these mate-rials is growing, but the ideal ones have not been found yet. The present review aims at exploring some applications of hybrid materials, with particular mention to urological and cardiovascular fields: in the first case, the efforts to find a construct that can guarantee impermeability, mechanical resistance and patency will be herein illustrated; in the second case, the search for impermeability, hemocompatibility and adequate compliance will be been disclosed.

Background Due to the shortage of organs’ donors that limits biological heart transplantations, mechanical circulatory supports can be implanted in case of refractory end-stage heart failure to replace partially (Ventricular Assist Device, VAD) or completely (Total Artificial Heart, TAH) the cardiac function. The hemocompatibility of mechanical circulatory supports is a fundamental issue that has not yet been fully matched; it mostly depends on the nature of blood-contacting surfaces. Methods In order to obtain hemocompatible materials, a pool of hybrid membranes was fabricated by coupling a synthetic polymer (polycarbonate urethane, commercially available in two formulations) with a decellularized biological tissue (porcine pericardium). To test their potential suitability as candidate materials for realizing the blood-contacting surfaces of a novel artificial heart, hybrid membranes have been preliminarily characterized in terms of physicochemical, structural and mechanical properties. Results Our results ascertained that the hybrid membranes are properly stratified, thus allowing to expose their biological side to blood and their polymeric surface to the actuation system of the intended device. From the biomechanical point of view, the hybrid membranes can withstand deformations up to more than 70 % and stresses up to around 8 MPa. Conclusions The hybrid membranes are suitable for the construction of the ventricular chambers of innovative mechanical circulatory support devices.

Over the years, several devices have been created (and the development of many others is currently in progress) to be in permanent contact with blood: mechanical circulatory supports represent an example thereof. The hemocompatibility of these devices largely depends on the chemical composition of blood-contacting components. In the present work, an innovative material (hybrid membrane) is proposed to fabricate the inner surfaces of a pulsatile ventricular chamber: it has been obtained by coupling a synthetic polymer (e.g., commercial polycarbonate urethane) with decellularized porcine pericardium. The hemocompatibility of the innovative material has been preliminarily assessed by measuring its capacity to promote thrombin generation and induce platelet activation. Our results demonstrated the blood compatibility of the proposed hybrid membrane.

Bladder cancer (BC) is among the most common malignancies in the world and a relevant cause of cancer mortality. BC is one of the most frequent causes for bladder removal through radical cystectomy, the gold-standard treatment for localized muscle-invasive and some cases of high-risk, non-muscle-invasive bladder cancer. In order to restore urinary functionality, an autologous intestinal segment has to be used to create a urinary diversion. However, several complications are associated with bowel-tract removal, affecting patients’ quality of life. The present study project aims to develop a bio-engineered material to simplify this surgical procedure, avoiding related surgical complications and improving patients’ quality of life. The main novelty of such a therapeutic approach is the decellularization of a porcine small intestinal submucosa (SIS) conduit to replace the autologous intestinal segment currently used as urinary diversion after radical cystectomy, while avoiding an immune rejection. Here, we performed a preliminary evaluation of this acellular product by developing a novel decellularization process based on an environmentally friendly, mild detergent, i.e., Tergitol, to replace the recently declared toxic Triton X-100. Treatment efficacy was evaluated through histology, DNA, hydroxyproline and elastin quantification, mechanical and insufflation tests, two-photon microscopy, FTIR analysis, and cytocompatibility tests. The optimized decellularization protocol is effective in removing cells, including DNA content, from the porcine SIS, while preserving the integrity of the extracellular matrix despite an increase in stiffness. An effective sterilization protocol was found, and cytocompatibility of treated SIS was demonstrated from day 1 to day 7, during which human fibroblasts were able to increase in number and strongly organize along tissue fibres. Taken together, this in vitro study suggests that SIS is a suitable candidate for use in urinary diversions in place of autologous intestinal segments, considering the optimal results of decellularization and cell proliferation. Further efforts should be undertaken in order to improve SIS conduit patency and impermeability to realize a future viable substitute.

The most common aortic valve diseases in adults are stenosis due to calcification and regurgitation. In pediatric patients, aortic pathologies are less common. When a native valve is surgically replaced by a prosthetic one, it is necessary to consider that the latter has a limited durability. In particular, current bioprosthetic valves have to be replaced after approximately 10 years; mechanical prostheses are more durable but require the administration of permanent anticoagulant therapy. With regard to pediatric patients, both mechanical and biological prosthetic valves have to be replaced due to their inability to follow patients’ growth. An alternative surgical substitute can be represented by the acellular porcine aortic valve that exhibits less immunogenic risk and a longer lifespan. In the present study, an efficient protocol for the removal of cells by using detergents, enzyme inhibitors, and hyper- and hypotonic shocks is reported. A new detergent (Tergitol) was applied to replace TX-100 with the aim to reduce toxicity and maximize ECM preservation. The structural integrity and efficient removal of cells and nuclear components were assessed by means of histology, immunofluorescence, and protein quantification; biomechanical properties were also checked by tensile tests. After decellularization, the acellular scaffold was sterilized with a standard protocol and repopulated with bone marrow mesenchymal stem cells to analyze its biocompatibility profile.

Total hip arthroplasty is one of the most common and successful orthopedic surgeries. Sometimes, periprosthetic osteolysis occurs associated with the stress-shielding effect: it results in the reduction of bone density, where the femur is not correctly loaded, and in the formation of denser bone, where stresses are confined. This paper illustrates the stress shielding effect as a cause of the failing replacement of the hip joint. An extensive literature survey has been accomplished to describe the phenomenon and identify solutions. The latter refer to the design criteria and the choice of innovative materials/treatments for prosthetic device production. Experimental studies and numerical simulations have been reviewed. The paper includes an introduction to explain the scope; a section illustrating the causes of the stress shielding effect; a section focusing on recent attempts to redefine prosthetic device design criteria, current strategies to improve the osteointegration process, and a number of innovative biomaterials; functionally graded materials are presented in a dedicated section: they allow customizing prosthesis features with respect to the host bone. Conclusions recommend an integrated approach for the production of new prosthetic devices: the “engineering community” has to support the “medical community” to assure an effective translation of research results into clinical practice.

Right ventricle outflow tract obstruction (RVOTO) is a congenital pathological condition that contributes to about 15% of congenital heart diseases. In most cases, the replacement of the right ventricle outflow in pediatric age requires subsequent pulmonary valve replacement in adulthood. The aim of this study was to investigate the extracellular matrix scaffold obtained by decellularization of the porcine pulmonary valve using a new detergent (Tergitol) instead of Triton X-100. The decellularized scaffold was evaluated for the integrity of its extracellular matrix (ECM) structure by testing for its biochemical and mechanical properties, and the cytotoxicity/cytocompatibility of decellularized tissue was assessed using bone marrow-derived mesenchymal stem cells. We concluded that Tergitol could remove the nuclear material efficiently while preserving the structural proteins of the matrix, but without an efficient removal of the alpha-gal antigenic epitope. Therefore, Tergitol can be used as an alternative detergent to replace the Triton X-100.

The covalent functionalization of synthetic peptides allows the modification of different biomaterials (metallic, polymeric, and ceramic), which are enriched with biologically active sequences to guide cell behavior. Recently, this strategy has also been applied to decellularized biological matrices. In this study, the covalent anchorage of a synthetic peptide (REDV) to a pericardial matrix decellularized via Schiff base is realized starting from concentrated peptide solutions (10−4 M and 10−3 M). The use of a labeled peptide demonstrated that as the concentration of the working solution increased, the surface density of the anchored peptide increased as well. These data are essential to pinpointing the concentration window in which the peptide promotes the desired cellular activity. The matrices were extensively characterized by Water Contact Angle (WCA) analysis, Differential Scanning Calorimetry (DSC) analysis, geometric feature evaluation, biomechanical tests, and preliminary in vitro bioassays.

The history of biomaterials dates back to the mists of time: human beings had always used exogenous materials to facilitate wound healing and try to restore damaged tissues and organs. Nowadays, a wide variety of materials are commercially available and many others are under investigation to both maintain and restore bodily functions. Emerging clinical needs forced the development of new biomaterials, and lately discovered biomaterials allowed for the performing of new clinical applications. The definition of biomaterials as materials specifically conceived for biomedical uses was raised when it was acknowledged that they have to possess a fundamental feature: biocompatibility. At first, biocompatibility was mainly associated with biologically inert substances; around the 1970s, bioactivity was first discovered and the definition of biomaterials was consequently extended. At present, it also includes biologically derived materials and biological tissues. The present work aims at walking across the history of biomaterials, looking towards the scientific literature published on this matter. Finally, some current applications of biomaterials are briefly depicted and their future exploitation is hypothesized.

The words "Translational" and "Medicine" have been recently coupled to indicate a combination of disciplines, resources, expertise, and techniques aimed at enhancing prevention, diagnosis, and therapies. As stated in 2015 by the European Society for Translational Medicine, translational medicine is "an interdisciplinary branch of the biomedical field supported by three main pillars: benchside, bedside and community". By definition, Translational Medicine is a highly interdisciplinary field, which gathers several specialties aimed at improving the global healthcare system.With regard to the assessment of the microcirculatory function, it is worthwhile to mention the growing interest from both basic research and clinical practice. Microcirculation is where the exchange of substances between blood and tissues takes place. Thus, it plays a key role in the pathophysiology of many diseases. Nonetheless, a gap does exist between the theoretical analysis of the microcirculatory function and its clinical exploitation. This gap can be due to the weak dissemination of analytical methods and theoretical results within the clinical community, which also delays the establishment of specific operative guidelines.This paper aims at encouraging, and possibly accelerating, the translation of basic research outcomes on microcirculatory function assessment into clinical applications.

(1) Background: shape, dimension, hemodynamics, and hemocompatibility are just a few of the several challenging key points that must be addressed in designing any suitable solution for the ventricular chamber of mechanical circulatory support devices. A preliminary evaluation of different geometries of bellow-like ventricular chambers is herein proposed. The chambers were made with a polycarbonate urethane that is acknowledged to be a hemocompatible polymer. (2) Methods: an explicit dynamic computational analysis was performed. The actuation of the three chambers was simulated without the presence of an internal fluid. Maximum stress and strain values were identified, as well as the most critical regions. Geometric changes were checked during simulated motion to verify that the dimensional constraints were satisfied. (3) Results: one chamber appeared to be the best solution compared to the others, since its dimensional variations were negligible, and effective stresses and strains did not reach critical values. (4) Conclusions: the identification of the best geometric solution will allow proceeding with further experimental studies. Fluid&ndash, structure interactions and fatigue analyses were investigated.

Xenogeneic pericardium-based substitutes are employed for several surgical indications after chemical shielding, limiting their biocompatibility and therapeutic durability. Adverse responses to these replacements might be prevented by tissue decellularization, ideally removing cells and preserving the original extracellular matrix (ECM). The aim of this study was to compare the mostly applied pericardia in clinics, i.e. bovine and porcine tissues, after their decellularization, and obtain new insights for their possible surgical use. Bovine and porcine pericardia were submitted to TRICOL decellularization, based on osmotic shock, detergents and nuclease treatment. TRICOL procedure resulted in being effective in cell removal and preservation of ECM architecture of both species&rsquo, scaffolds. Collagen and elastin were retained but glycosaminoglycans were reduced, significantly for bovine scaffolds. Tissue hydration was varied by decellularization, with a rise for bovine pericardia and a decrease for porcine ones. TRICOL significantly increased porcine pericardial thickness, while a non-significant reduction was observed for the bovine counterpart. The protein secondary structure and thermal denaturation profile of both species&rsquo, scaffolds were unaltered. Both pericardial tissues showed augmented biomechanical compliance after decellularization. The ECM bioactivity of bovine and porcine pericardia was unaffected by decellularization, sustaining viability and proliferation of human mesenchymal stem cells and endothelial cells. In conclusion, decellularized bovine and porcine pericardia demonstrate possessing the characteristics that are suitable for the creation of novel scaffolds for reconstruction or replacement: differences in water content, thickness and glycosaminoglycans might influence some of their biomechanical properties and, hence, their indication for surgical use.

The wavelet analysis has been applied to the Laser Doppler Fluxmetry for assessing the frequency spectrum of the flowmotion to study the microvascular function waves.Although the application of wavelet analysis has allowed a detailed evaluation of the microvascular function, its use does not seem to be yet widespread over the last two decades.Aiming to improve the diffusion of this methodology, we herein present a systematic review of the literature about the application of the wavelet analysis to the laser Doppler fluxmetry signal. A computer research has been performed on PubMed and Scopus databases from January 1990 to December 2017. The used terms for the investigation have been "wavelet analysis", "wavelet transform analysis", "Morlet wavelet transform" along with the terms "laser Doppler", "laserdoppler" and/or "flowmetry" or "fluxmetry". One hundred and eighteen studies have been found. After the scrutiny, 97 studies reporting data on humans have been selected. Fifty-three studies, 54.0% (95% CI 44.2-63.6) pooled rate, have been performed on 892 healthy subjects and 44, 45,9 % (95% CI 36.3-55.7%) pooled rate have been performed on 1679 patients. No significant difference has been found between the two groups (p 0,81). On average, the number of studies published each year was 4.8 (95% CI 3.4-6.2). The trend of studies production has increased significantly from 1998 to 2017, (p 0.0006). But only the studies on patients have shown a significant increase trend along the years (p 0.0003), than the studies on healthy subjects (p 0.09).In conclusion, this review highlights that despite being a promising and interesting methodology for the study of the microcirculatory function, the wavelet analysis has remained still neglected.

Decellularized xenogeneic scaffolds have shown promise to be employed as compatible and functional cardiovascular biomaterials. However, one of the main barriers to their clinical exploitation is the lack of appropriate sterilization procedures. This study investigated the efficiency of a two-step sterilization method, antibiotics/antimycotic (AA) cocktail and peracetic acid (PAA), on porcine and bovine decellularized pericardium. In order to assess the efficiency of the method, a sterilization assessment protocol was specifically designed, comprising: i) controlled contamination with a known amount of bacteria; ii) sterility test; iii) identification of contaminants through MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) mass spectrometry and iv) quantification by the Most Probable Number (MPN) method. This sterilization assessment protocol proved to be a successful tool to monitor and optimize the proposed sterilization method. The treatment with AA + PAA method provided sterile scaffolds while preserving the structural integrity and biocompatibility of the decellularized porcine and bovine tissues. However, surface properties and cellular adhesion resulted slightly impaired on porcine pericardium. This work developed a sterilization method suitable for decellularized pericardial scaffolds that could be adopted for in vivo tissue engineering. Together with the proposed sterilization assessment protocol, this decontamination method will foster the clinical translation of decellularized xenogeneic substitutes. Statement of Significance Clinical application of functional and compatible xenogeneic decellularized scaffolds has been delayed due to the lack of appropriate sterilization methodologies. In this study, it was investigated an effective sterilization method optimized for porcine and bovine decellularized pericardia, based on the use of antibiotics/antimycotics followed by peracetic acid treatment. This treatment effectively sterilizes both species scaffolds, proves to maintain tissue overall structure and components, preserves biocompatibility and biomechanical properties. Furthermore, it was also developed a sterilization assessment protocol used to monitor and validate the previous method, consisting in three main parts: i) controlled contamination; ii) sterility test, and iii) identification and quantification of contaminants. Both methodologies were optimized for the tissues in study but can be applied to other scaffolds and accelerate their clinical translation.

Bovine and porcine pericardia are currently used for manufacturing prosthetic heart valves: their design has become an increasingly important area of investigation in parallel with progressively expanding indications for the transcutaneous approach to heart valves replacement. Before being cut and shaped, pericardial tissues are expected to be properly characterized. Actually, the mechanical assessment of these biomaterials lacks standardized protocols. In particular, the role of preconditioning for achieving a constant mechanical response of tissue samples is still controversial. In the present work, the mechanical response to uniaxial load of native bovine and porcine pericardia, with and without preconditioning was assessed; moreover, the mechanical behavior of pericardia was investigated and explained. It was demonstrated that: (i) pericardial tissue samples hold memory of the loading history but just within the extent of the deformation applied; (ii) the behavior of native bovine and porcine pericardia in response to load is explained by a mechanism based on the additive recruitment of collagen fibers; (iii) the current concept that plasticity is absent in pericardium has to be at least in part reconsidered.

The formation of thrombotic deposits affects the functionality of mechanical prosthetic heart valves; as a consequence, mechanical valves thrombosis needs early diagnosis to prevent thromboembolic events. This paper compares the acoustic signals produced by two commercial bileaflet mechanical heart valves in the closing phase to detect the presence of thrombi. The closing sounds were recorded in vitro by means of a phonocardiographic device under different hydrodynamic conditions. Thrombotic deposits of different weight and shape were applied onto the valve leaflet and the annular housing, until the movement of one leaflet was completely blocked. From the acoustic signals, the corresponding spectra were calculated and four diagnostic frequency bands were identified: their comparison allowed detecting malfunctioning valves because of the presence of thrombotic formations.

There are limited therapeutic options for final treatment of end-stage heart failure. Among them, implantation of a total artificial heart (TAH) is an acceptable strategy when suitable donors are not available. TAH development began in the 1930s, followed by a dramatic evolution of the actuation mechanisms operating the mechanical pumps. Nevertheless, the performance of TAHs has not yet been optimized, mainly because of the low biocompatibility of the blood-contacting surfaces. Low hemocompatibility, calcification, and sensitivity to infections seriously affect the success of TAHs. These unsolved issues have led to the withdrawal of many prototypes during preclinical phases of testing. This review offers a comprehensive analysis of the pathophysiological events that may occur in the materials that compose TAHs developed to date. In addition, this review illustrates bioengineering strategies to prevent these events and describes the most significant steps toward the achievement of a fully biocompatible TAH.

Bovine and porcine pericardia are currently used for manufacturing prosthetic heart valves: their design has become an increasingly important area of investigation in parallel with progressively expanding indications for the transcutaneous approach to heart valves replacement. Before being cut and shaped, pericardial tissues are expected to be properly characterized. Actually, the mechanical assessment of these biomaterials lacks standardized protocols. In particular, the role of preconditioning for achieving a constant mechanical response of tissue samples is still controversial. In the present work, the mechanical response to uniaxial load of native bovine and porcine pericardia, with and without preconditioning was assessed; moreover, the mechanical behavior of pericardia was investigated and explained. It was demonstrated that: (i) pericardial tissue samples hold memory of the loading history but just within the extent of the deformation applied; (ii) the behavior of native bovine and porcine pericardia in response to load is explained by a mechanism based on the additive recruitment of collagen fibers; (iii) the current concept that plasticity is absent in pericardium has to be at least in part reconsidered.

Autologous platelet-rich hemocomponents have emerged as potential biologic tools for regenerative purpose, but their therapeutic efficacy still remains controversial. This work represents the characterization study of an innovative autologous leukocyte-fibrin-platelet membrane (LFPm), which we prepared according to a novel protocol involving multiple cycles of apheresis. The high content in fibrinogen gave to our hemocomponent the appearance of a manipulable and suturable membrane with high elasticity and deformation capacity. Moreover, being highly enriched with platelets, leukocytes, and monocytes/macrophages, the LFPm sustained the local release of bioactive molecules (platelet derived growth factor, vascular endothelial growth factor, interleukin-10, and tumour necrosis factor alpha). In parallel, the evaluation of stemness potential highlighted also that the LFPm contained cells expressing pluripotency and multipotency markers both at the messenger ribonucleic acid (NANOG, SOX2, THY1, NT5E, and ENG) and surface-protein level (CD44high /CD73+ /CD34+ /CD117+ /CD31+ ). Finally, biodegradation analysis interestingly showed a good stability of the membrane for at least 3 weeks in vitro and 1 week in vivo. In both cases, biodegradation was associated with progressive exposure of fibrin scaffold, loss/migration of cellular elements, and release of growth factors. Overall, collected evidence could shed some light on the regenerative effect that LFPms may exert after the autologous implant on a defect site.

The skin flowmotion of 13 patients suffering from critical limb ischemia (CLI) was studied with wavelet analysis (WA) of the laser Doppler signals (LDS). The WA selects six different frequency components (FCs), each relating to a specific cardiovascular system structures activities; FC I 1-2 Hz heart, FC II 0.2 Hz respiratory, FC III 0.1 Hz myogenic, FC IV 0.04 Hz, sympathetic, FC V 0.01 Hz, and FC VI 0.007 Hz endothelial. The aim of the study was to observe which FC changed after the limb revascularization. The LDS was measured at the dorsum of the foot, one week before and no later than 30 days after revascularisation. The absolute and relative amplitude and energy of the flowmotion WA FCs, the ankle brachial pressure index (ABI) and the transcutaneous pressure of oxygen (TcpO2) were assessed before and after revascularization. The results showed that after successful revascularization ABI and TcpO2 increased from 0.34 ± 0.10 to 0.54 ± 0.09 (p 0.0003) and from 20.3 ± 13.4 to 43.8 ± 18.7 mmHg (p 0.0002) whereas only the absolute amplitude and energy of the cardiac FC I increased from 0.57 ± 0.44 to 1.07 ± 0.69 (P 0.002) AU and 1.14 ± 1.78 AU2 to 3.54 ± 3.78 AU2 (p 0.004). In conclusion after limb revascularization the cardiac component of the flowmotion increased maybe because the cardiac stroke volume had more influence over the skin arterioles.

The haemodynamics of skin microcirculation can be quantitatively evaluated by Laser Doppler Fluxmetry (LDF). LDF signal in human skin shows periodic oscillations. Spectral analysis by wavelet transform displays six characteristic frequency intervals (FI) from 0.005 to 2 Hz, related to distinct vascular structures activities: heart (0.6-2 Hz), sympathetic respiratory (0.145-0.6 Hz), myogenic (0.052-0.145 Hz), local sympathetic nerve (0.021-0.052 Hz) and endothelial cells NO dependent (0.0095-0.021 Hz) and NO independent (0.005-0.0095 Hz). The most advanced stage of peripheral arterial obstructive disease is the critical limb ischemia (CLI), which causes the reduction of blood perfusion threatening limb viability. Besides macrocirculatory alterations, many studies have shown microvascular misdistribution of skin blood flow as the main factor that leads patients to CLI. Revascularization can save limb and patient's life, too. In the present study, LDF signals have been recorded on the skin of the foot dorsum in 15 patients suffering from CLI. LDF signals have been analyzed before and after limb revascularization by means of the wavelet analysis. Significant changes in frequency distribution before and after limb revascularization have been detected: the median normalized values of spectral power increases for 49.8% (p = 0.0341) in the frequency range 0.050328-0.053707 Hz, whereas spectral power decreases for 77.1% (p = 0.0179) in the frequency range 0.018988-0.029284 Hz. We can conclude that changes in the frequency intervals occur after revascularization, shifting from a prevailing endothelial activity toward a prevailing sympathetic activity.

The hemodynamics of skin microcirculation can be clinically assessed by means of Laser Doppler Fluxmetry. Laser Doppler signals show periodic oscillations because of fluctuations of microvascular perfusion (flowmotion), which are sustained by contractions and relaxations of arteriolar walls rhythmically changing vessels diameter (vasomotion). The wavelet analysis applied to Laser Doppler signals displays six characteristic frequency intervals, from 0.005 to 2 Hz. Each interval is assigned to a specific structure of the cardiovascular system: heart, respiration, vascular myocites, sympathetic terminations, and endothelial cells (dependent and independent on nitric oxide). Therefore, mechanisms of skin perfusion can be investigated through wavelet analysis. In the present work, examples of methods and results of wavelet analysis applied to Laser Doppler signals are reported. Laser Doppler signals were acquired in two groups of patients to check possible changes in vascular activities, before and after occlusive reactive hyperaemia, and before and after revascularization.

Haemophilic arthropathy is the major cause of disability in patients with haemophilia and, despite prophylaxis with coagulation factor concentrates, some patients still develop articular complications. We evaluate the feasibility of a tissue engineering approach to improve current clinical strategies for cartilage regeneration in haemophiliacs by using autologous chondrocytes (haemophilic chondrocytes; HaeCs). Little is known about articular chondrocytes from haemophilic patients and no characterisation has as yet been performed. An investigation into whether blood exposure alters HaeCs should be interesting from the perspective of autologous implants. The typical morphology and expression of specific target genes and surface markers were therefore assessed by optical microscopy, reverse transcription plus the polymerase chain reaction (PCR), real-time PCR and flow-cytometry. We then considered chondrocyte behaviour on a bio-hybrid scaffold (based on polyvinyl alcohol/Wharton's jelly) as an in vitro model of articular cartilage prosthesis. Articular chondrocytes from non-haemophilic donors were used as controls. HaeC morphology and the resulting immunophenotype CD44(+)/CD49c(+)/CD49e(+)/CD151(+)/CD73(+)/CD49f(-)/CD26(-) resembled those of healthy donors. Moreover, HaeCs were active in the transcription of genes involved in the synthesis of the extracellular matrix proteins of the articular cartilage (ACAN, COL1A, COL2A, COL10A, COL9A, COMP, HAS1, SOX9), although the over-expression of COL1A1, COL10A1, COMP and HAS was observed. In parallel, the composite scaffold showed adequate mechanical and biological properties for cartilage tissue engineering, promoting chondrocyte proliferation. Our preliminary evidence contributes to the characterisation of HaeCs, highlighting the opportunity of using them for autologous cartilage implants in patients with haemophilia.

This work elucidates the mechanisms of skin microcirculation response to local heating at 44°C in vasculopathic patients. Laser Doppler and tcpO2 were simultaneously acquired. Patients were selected on the basis of tcpO2: Group A30 mmHg; Group B 30-50 mmHg; Group C50 mmHg. The wavelet analysis of signal oscillations displays six frequency intervals. Each interval is assigned to a specific cardiovascular activity. The contributions of cardiac, myogenic and neurogenic activities were selectively detected. Thermal stimulation increased relative amplitude in all patients: heart activity by +103.26% in A, +162.84% in B, +454.54% in C; myogenic activity by +52.45% in A, +38.51% in B, +156.19% in C; neurogenic activity +43.36% in A, +74.15% in B, +242.42% in C. Thermal stimulation increased relative power in all patients: heart activity by +365.30% in A, +473.72% in B, +1393.77% in C; myogenic activity by +106.92% in A, +66.03% in B, +380.18% in C; neurogenic activity by +77.00% in A, +162.65% in B, +771.93% in C.This work demonstrates that the spectral analysis allows extracting from Laser Doppler signals more information than that can be gained by solely investigating perfusion values over time.

The development of viable and functional tissue-engineered heart valves (TEHVs) is a challenge that, for almost two decades, the scientific community has been committed to face to create life-lasting prosthetic devices for treating heart valve diseases. One of the main drawbacks of tissue-based commercial substitutes, xenografts and homografts, is their lack of viability, and hence failure to grow, repair, and remodel. In adults, the average bioprostheses life span is around 13 years, followed by structural valve degeneration, such as calcification; in pediatric, mechanical valves are commonly used instead of biological substitutes, as in young patients, the mobilization of calcium, due to bone remodeling, accelerates the calcification process. Moreover, neither mechanical nor bioprostheses are able to follow children's body growth. Cell seeding and repopulation of acellular heart valve scaffolds, biological and polymeric, appears as a promising way to create a living valve. Biomechanical stimuli have significant impact on cell behavior including in vitro differentiation, and physiological hemodynamic conditioning has been found to promote new tissue development. These concepts have led scientists to design bioreactors to mimic the in vivo environment of heart valves. Many different types of somatic and stem cells have been tested for colonizing both the surface and the core of the valve matrix but controversial results have been achieved so far.

Heart valve disorders, caused by congenital defects, rheumatic fever, calcification, myocardial infarction and other cardiovascular diseases, often require native valves to be replaced by bio-prosthetic devices or mechanical heart valves (MHVs). Among MHVs, bileaflet valves are usually preferred for their hemodynamic features, similar to physiological ones, and their durability, but they are prone to complications due to thromboembolic events. Due to the asynchronous closure of the leaflets, bileaflet MHVs are also known to produce closing sounds typically characterized by the presence of two peaks in the time domain. The detection of this “double click” in the signal may be useful for the early diagnosis of bileaflet MHV malfunction. The closing sound is actually a non-stationary signal that can be properly explored by means of time-frequency analysis. This paper describes a preliminary approach to the investigation of bileaflet MHV closing sounds performed by Continuous Wavelet Transform (CWT) analysis. Signals were collected from 3 patients immediately after surgery by means of the Myotis 3C, which is a traditional phonocardiographic apparatus. Signals were analyzed by two algorithms: one embedded in the Myotis 3C, based on the Fast Fourier Transform (FFT); and one specifically created for the purposes of the present study, based on CWT. The performance of these algorithms was compared and the results showed that the proposed CWT technique correctly classifies as “double” a large number of clicks that are recognized as “single” by the Myotis 3C.

Investigations on the relationships between the properties of biomaterial surfaces and cell adhesion/proliferation processes have recently gained increasing interest. To describe the behaviour of cells adhering and proliferating over different types of (and/or differently treated) substrates, some mathematical models have been also suggested in literature; these models consider both the dependence of cell adhesion/proliferation over time, and the influence of substrate morphology in allowing (or even hampering) cell attachment. Major developments in the biochemical functionalization of the materials used for the production of endosseous devices have been achieved; the ability of the so-called "biomimetic" surfaces to promote cell adhesion, thus favoring the osseointegration process, is already well acknowledged. The aim of this study was to formulate a mathematical model for osteoblast adhesion, mediated by an adhesion peptide (sequence 351-359 mapped on the Human Vitronectin Protein) covalently grafted to a titanium-based surface. To assure a highly homogenous orientation of the peptide to cells, the "specific functionalization" strategy was properly designed. Enzymatic detachment assays allowed comparing osteoblast behaviour over three differently treated titanium substrates (i.e., oxidized, silanized, and peptide-grafted), thus determining how and how much the bioactive peptide can improve the strength of cell adhesion. The results confirmed the capacity of the peptide to increase cell adhesion and adhesion strength; moreover, the role of the peptide was described by a mathematical equation characterizing cells behaviour.

Mammalian pericardia are currently used for the production of percutaneous prosthetic heart valves. The characteristics of biological tissues largely influence the durability of prosthetic devices used in the percutaneous approach and in traditional surgery, too. This paper reviews methodologies employed to assess and compare mechanical properties of pericardial patches from different mammalian species in order to identify the biomaterials adequate for manufacturing prosthetic heart valves.

Laser Doppler Fluxmetry is used to evaluate the hemodynamics of skin microcirculation. Laser Doppler signals contain oscillations due to fluctuations of microvascular perfusion. By performing spectral analysis, six frequency intervals from 0.005 to 2 Hz have been identified and assigned to distinct cardiovascular structures: heart, respiration, vascular myocites, sympathetic terminations and endothelial cells (dependent and independent on nitric oxide). Transcutaneous electrical pulses are currently applied to treat several diseases, i.e. neuropathies and chronic painful leg ulcers. Recently, FREMS (Frequency Rhythmic Electrical Modulation System) has been applied to vasculopathic patients, too. In this study Laser Doppler signals of skin microcirculation were measured in five patients with intermittent claudication, before and after the FREMS therapy. Changes in vascular activities were assessed by wavelet transform analysis. Preliminary results demonstrate that FREMS induces alterations in vascular activities.

A fundamental goal in the field of implantology is the design of specific devices able to induce a controlled and rapid “osseointegration”. This result has been achieved by means of surface modifications aimed at optimizing implant-to-bone contact; furthermore, bone cell adhesion on implant surface has been directly improved by the application of biomolecules that stimulate new tissue formation, thus controlling interactions between biological environment and implanted materials. Actually, methods for biochemical factor delivery at the interface between implant surface and biological tissues are under investigation; a reliable technique is represented by the inclusion of biologically active molecules into biocompatible and biodegradable materials used for coating implant surface. This paper focuses the application of three polymeric materials already acknowledged in the clinical practice, i.e. poly- l -lactic acid (PLLA), poly- dl -lactic acid (PDLA), and sodium alginate hydrogel. They have been used to coat Ti (Ti2) and Ti6Al4V (Ti5) disks; their characteristics have been determined and their performances compared, with specific regard to the ability in allowing osteoblast adhesion in vitro. Moreover, profilometry data analysis permitted to identify a specific roughness parameter (peak density) which mainly controls the amount of osteoblast adhesion.

Notwithstanding their wide exploitation, biological prosthetic heart valves are characterized by limited durability (10–15 years). The treatment of biological tissues with chemical crosslinking agents such as glutaraldehyde accounts for the enhanced risk of structural deterioration associated with the early failure of bioprosthetic valves. To overcome the shortcomings of the currently available solutions, adoption of decellularized biological tissues of animal origin has emerged as a promising approach. The present study aims to assess in vitro cardiovascular scaffolds composed of bovine pericardium decellularized with the novel TRITDOC (TRIton-X100 and TauroDeOxyCholic acid) procedure. The effects of the treatment have been assessed by means of histological, biomolecular, cellular, biochemical and biomechanical analyses. The TRITDOC procedure grants the complete decellularization of bovine pericardial scaffolds while preserving the extracellular matrix architecture and the biomechanical properties. With a dedicated ELISA test, the TRITDOC procedure has been proven to ensure the complete removal of the alphaGal antigen, responsible for hyperacute rejection and for long-term deterioration of xenogenic biomaterials. Static seeding of the acellular pericardial patches with human adipose-derived stem cells resulted in an evenly repopulated scaffold without signs of calcification. The in vitro cyto-/immuno-compatibility response of the TRITDOC-bovine pericardium was compared with glutaraldehyde-treated xenogenic pericardium collected from two bioprosthetic devices currently used in clinical practice: PERIMOUNT MAGNA and TRIFECTATM. TRITDOC-bovine pericardium exhibited lower complement activation, lower cytotoxicity and a lower tendency to secrete pro-inflammatory cytokines compared to the tested commercial bioprostheses. Therefore, TRITDOC-decellularized pericardium could be considered as possible candidate material for the production of prosthetic heart valves.

The features of implant devices and the reactions of bone-derived cells to foreign surfaces determine implant success during osseointegration. In an attempt to better understand the mechanisms underlying osteoblasts attachment and spreading, in this study adhesive peptides containing the fibronectin sequence motif for integrin binding (Arg-Gly-Asp, RGD) or mapping the human vitronectin protein (HVP) were grafted on glass and titanium surfaces with or without chemically induced controlled immobilization. As shown by total internal reflection fluorescence microscopy, human osteoblasts develop adhesion patches only on specifically immobilized peptides. Indeed, cells quickly develop focal adhesions on RGD-grafted surfaces, while HVP peptide promotes filopodia, structures involved in cellular spreading. As indicated by immunocytochemistry and quantitative polymerase chain reaction, focal adhesions kinase activation is delayed on HVP peptides with respect to RGD while an osteogenic phenotypic response appears within 24 h on osteoblasts cultured on both peptides. Cellular pathways underlying osteoblasts attachment are, however, different. As demonstrated by adhesion blocking assays, integrins are mainly involved in osteoblast adhesion to RGD peptide, while HVP selects osteoblasts for attachment through proteoglycan-mediated interactions. Thus an interfacial layer of an endosseous device grafted with specifically immobilized HVP peptide not only selects the attachment and supports differentiation of osteoblasts but also promotes cellular migration.