Can Heart Valve Decellularization Be Standardized? A Review of the Parameters Used for the Quality Control of Decellularization Processes.

When a tissue or an organ is considered, the attention inevitably falls on the complex and delicate mechanisms regulating the correct interaction of billions of cells that populate it. However, the most critical component for the functionality of specific tissue or organ is not the cell, but the cell-secreted three-dimensional structure known as the extracellular matrix (ECM). Without the presence of an adequate ECM, there would be no optimal support and stimuli for the cellular component to replicate, communicate and interact properly, thus compromising cell dynamics and behaviour and contributing to the loss of tissue-specific cellular phenotype and functions. The limitations of the current bioprosthetic implantable medical devices have led researchers to explore tissue engineering constructs, predominantly using animal tissues as a potentially unlimited source of materials. The high homology of the protein sequences that compose the mammalian ECM, can be exploited to convert a soft animal tissue into a human autologous functional and long-lasting prosthesis ensuring the viability of the cells and maintaining the proper biomechanical function. Decellularization has been shown to be a highly promising technique to generate tissue-specific ECM-derived products for multiple applications, although it might comprise very complex processes that involve the simultaneous use of chemical, biochemical, physical and enzymatic protocols. Several different approaches have been reported in the literature for the treatment of bone, cartilage, adipose, dermal, neural and cardiovascular tissues, as well as skeletal muscle, tendons and gastrointestinal tract matrices. However, most of these reports refer to experimental data. This paper reviews the most common and latest decellularization approaches that have been adopted in cardiovascular tissue engineering. The efficacy of cells removal was specifically reviewed and discussed, together with the parameters that could be used as quality control markers for the evaluation of the effectiveness of decellularization and tissue biocompatibility. The purpose was to provide a panel of parameters that can be shared and taken into consideration by the scientific community to achieve more efficient, comparable, and reliable experimental research results and a faster technology transfer to the market.

Wet-priming extracorporeal membrane oxygenation device maintains sterility for up to 35 days of follow-up.

In emergency cases, rapid extracorporeal membrane oxygenation (ECMO) device initialization is able to drastically reduce the incidence of patient morbidity and/or mortality. Pre-assembled and ready-to-use ECMO circuits might save up to 30-60 critical minutes in patient management. Six ECMO circuits (Oxygenator D905 EOS with REVOLUTION™ pump and Sorin PTS) were assembled in the operating room in standard conditions and then placed at 37°C for 35 days in order to evaluate possible contamination and ingrowth of micro-organisms. Every 7 days after ECMO circuit assembly and wet-priming, samples of priming fluid were analyzed to verify the presence/absence of possible common contaminants (Enterobacteriaceae, Staphylococcus aureus and fungi). Moreover, two supplementary circuits, used as positive controls, were deliberately inoculated with a known concentration of a Escherichia coli strain and prime samplings carried out at different time-points to determine bacterial growth rate. Sterility was maintained in the ECMO circuits for up to 35 days.

[Functional evaluation in patients with pulmonary tuberculosis sequelae]. / Avaliação funcional em pacientes com sequela pulmonar de tuberculose.

INTRODUCTION: In pulmonary tuberculosis, the presence of extensive residual lung lesions can be a predictor of permanent disability due to respiratory failure. OBJECTIVE: To compare functional and respiratory changes in patients with pulmonary tuberculosis sequel who have completed treatment. METHOD: The study included patients who completed treatment within a period of 6 months (group I) and multidrug-resistant pulmonary tuberculosis patients who completed treatments of longer duration after the failure of the initial treatment (group II). We evaluated lung function by spirometry (Microlab ML 3500), the strength of respiratory muscles through the manovacuometry (MEP-maximal expiratory pressure and MIP- maximal inspiratory pressure) and the distance walked during the 6-minute walk (6MWT). RESULTS: 27 patients were included, 12 of whom belonged to group II, multidrug-resistant tuberculosis (MDRTB). Severe combined respiratory disorder was the most prevalent problem in group II of MDRTB; it was present in 9 patients. The MDRTB group (group II) showed significantly lower values when compared to Group I in FVC (72.06±14.95 vs 43.58±16.03% predicted), FEV1 (66.13±19.87 vs 33.08±15.64% predicted), MIP (68.40±22.78 vs 49.58±12.55 cmH(2)O), MEP (87.20±27.30 vs 59.08±12.23 cmH(2)O) and distance covered in 6MWT (484.21±74.01 vs 334.75±104.07 meters). CONCLUSION: Patients with multidrug resistant pulmonary tuberculosis who have undergone multiple treatments have more severe respiratory and functional impairment than patients who have had just a single treatment.

First quantitative assay of alpha-Gal in soft tissues: presence and distribution of the epitope before and after cell removal from xenogeneic heart valves.

Decellularized xenograft heart valves might be the ideal scaffolds for tissue engineered heart valves as the alternative to the currently used biological and mechanical prostheses. However, removal of the alpha-Gal epitope is a prerequisite to avoid hyperacute rejection of untreated xenograft material. The aim of this study was to develop an ELISA soft-tissue assay for alpha-Gal quantification in xenograft heart valves before and after a detergent-based (TriCol) or equivalent cell removal procedure. Leaflets from porcine valves were enzymatically digested to expose the epitope and reacted with the alpha-Gal monoclonal antibody M86 for its recognition. Rabbit erythrocytes were used as a reference for the quantification of alpha-Gal. Native aortic and pulmonary leaflets exhibited different epitope concentration: 4.33×10(11) vs. 7.12×10(11)/10 mg wet tissue (p<0.0001). Sampling of selected zones in native valves revealed a different alpha-Gal distribution within and among different leaflets. The pattern was consistent with immunofluorescence analysis and was unrelated to microvessel density distribution. After TriCol treatment alpha-Gal was no longer detectable in both pulmonary and aortic decellularized valves, confirming the ability of this method to remove both cells and alpha-Gal antigen. These results hold promise for a reliable quantitative evaluation of alpha-Gal in decellularized valves obtained from xenograft material for tissues engineering purposes. Additionally, this method is applicable to further evaluate currently used xenograft bioprostheses.

High third-order nonlinear optical susceptibility in new fluorinated poly(p-phenylenevinylene) copolymers measured with the Z-scan technique.

The third-order nonlinear optical properties of a series of copoly(2, 3, 5, 6-tetrafluoro-1, 4-phenylenevinylene-2, 5-dioctyloxy-1, 4-phenylenevinylene) that contain variable ratios of two differently substituted monomers have been studied in chloroform solutions at l=1064 nm by the picosecond Z-scan technique. Nonlinear refractive index n(2) of the samples investigated has been found to be negative, and a strong dependence of its magnitude on the copolymer's composition has been observed. The highest third-order nonlinear optical susceptibility, |x((3))|=(6 +/- 2)x 10(-10) esu, was measured for a copolymer obtained by reaction of equimolar quantitites of the parent monomers.

A synthetic strategy leading to monodisperse PPV oligomers by coupling reactions of vinyltrimethylsilanes.

A novel strategy for the synthesis of well-defined oligo(phenylenevinylene)s was developed. The procedure is entirely based upon two coupling processes, both involving vinyltrimethylsilanes. Bis(styryl)benzenes 2a-g bearing two octyloxy groups in the central aromatic ring and various substituents on the external aromatic rings were prepared in good yield by a regio- and stereoselective coupling reaction of 1 with different arenediazonium tetrafluoroborates. Oligomers with a more extended conjugated system, 4a-c, and with m-phenylene subunits 13a,b, were also readily obtained by conversion of the unsaturated trimethylsilyl derivatives 3a,c,d to the corresponding boron derivatives and a subsequent coupling reaction with compounds 2a and 2c.

Synthesis and characterization of poly(2,3,5,6-tetrafluoro-1,4-phenylenevinylene).

Poly(2,3,5,6-tetrafluoro-1,4-phenylenevinylene) (PTFPV) was prepared for the first time by the Stille cross-coupling reaction and the resulting material was characterized through MALDI-TOF mass spectrometry, employing a novel sample preparation protocol suitable for insoluble compounds; preliminary optical and electrooptical measurements were performed.

A simple and rapid nonisotopic method for sizing CAG repeats in the SCA1 gene.

Spinocerebellar ataxia type 1 is caused by the expansion of a CAG trinucleotide repeat, located at the 5' end of the gene responsible for the disease (SCA1 gene). We propose a simple and rapid method for SCA1 diagnosis, avoiding both radioactive and Southern blotting analysis. The method allows an accurate allele sizing by visualization of polymerase chain reaction products through a silver nitrate-stained polyacrylamide gel.

Nonisotopic method for accurate detection of (CAG)n repeats causing Huntington disease.

Huntington disease (HD) is a neurodegenerative disorder caused by an expanded trinucleotide repeat (CAG)n located at the 5' end of the novel IT15 gene. Discovery of this expansion allows the molecular diagnosis of HD by measuring repeat length. We applied a simple nonisotopic method to detect (CAG)n repeats, avoiding both radioactive and Southern transfer analysis. The assay is based on direct visualization of electrophoresed PCR products, after silver nitrate gel staining. Its accurate sizing of HD alleles allows presymptomatic diagnosis of at-risk persons. By avoiding isotopic manipulations, the method is safe and accurate, with no radioactive background bands. Furthermore, because it permits direct allele visualization after gel staining, the method is simple and rapid, allowing allele sizing within hours rather than days.

Genetic fitness in Huntington's Disease and Spinocerebellar Ataxia 1: a population genetics model for CAG repeat expansions.

An analysis of genetic fitness was performed in Huntington's Disease (HD) and Spinocerebellar Ataxia 1 (SCA1) families. Two partially overlapping samples were used: clinically defined HD and SCA1 patients from families ascertained in definite geographical areas, and molecularly typed carriers of HD and SCA1 mutations (CAG trinucleotide expansions). In both cases, a control group of normal relatives was used. HD and SCA1 patients born before 1915-20 had more children than normal controls. Carriers of HD and SCA1 mutations, all in the low/medium expansion range (37-49 and 47-54 CAG repeats respectively), had a higher number of children than controls up to more recent times (1935-1950). The reproduction of heterozygotes for large expansions could be analysed only in subjects born after 1950 and provided indirect evidence of a lower than normal number of children. The above results fit a model based on a differential fitness according to the degree of expansion. Such a model predicts that 1) up to relatively recently the frequency of alleles in the low/medium range has been maintained or even increased by the increased fitness of their carriers, as well as by new mutations, and 2) the frequency of large expansions, part of which are lost at each generation, is maintained through further expansions of alleles in the low/medium expansion range. The implications of such a model on linkage disequilibrium and the possible spread of these diseases in future generations are discussed.

Clinical study of large kindreds with autosomal dominant HLA-linked spinocerebellar ataxia (SCA1) of late onset.

Six families with SCA1 were studied. The clinical data on 35 patients are reported. Cerebellar and pyramidal system involvement was invariably found in association with brainstem, spinal cord and/or peripheral nervous system disorders. In our patients the clinical features appeared concordant when the patients with the same disease duration were compared. Previous reports of SCA1 families had shown great variability in clinical phenotype both interfamilial and intrafamilial. We suggest that the phenotype might appear more homogeneous if disease duration is taken into account.

Endurance training in the elderly nursing home patient.

Endurance training in nursing home patients was evaluated by selecting 15 of 150 patients for a conditioning program eliminating those patients with dementia and/or significant cardiac disease. After a medical and laboratory screen, all had a symptom-limited exercise test (SLXT) using arm and leg ergometry. Only two patients were able to use the treadmill and many had difficulty with leg cycling. Exercise capacities averaged 2-3METs in most patients. The exercise (E) group included eight randomly selected subjects who participated in a three times weekly upper and lower conditioning program using target heart rates based on the SLXT. The control (C) group continued in the daily nursing home routine. The SLXT was repeated at the end of one year. Two patients in each group expired from unassociated medical problems. Three patients in group E were hospitalized for brief periods. In group E there was a small but significant training effect in the arms (p = 0.05) but not in the legs. It would appear that a small training effect can occur in elderly nursing home patients with a conditioning program. The magnitude is limited by the low intensity of the program as well as the detraining effect of intercurrent illness. Functional limitations of the legs may be responsible for the lack of a training effect in these muscle groups.

Marked bone spur formation in a burn amputee patient.

This report presents an unusual case of a lower extremity burn amputee with a marked degree of bone spur formation. A 17-year-old man suffered 56% body surface area mixed-depth electrical and flame burns, necessitating left below knee amputation. He was admitted to a rehabilitation center 3 months postinjury for pylon fitting and gait training. Difficulty was encountered with poor skin tolerance to weight bearing because of the prominent distal bony margins in the stump. X-rays of the stump revealed a marked degree of linear bone spur formation, extending longitudinally from the distal tibia and fibula with multiple cross-bridges. The spur formation was considered an extensive bony exostosis of unclear etiology. Surgical revision was elected to obtain a stump more suitable for prosthetic tolerance, and to avoid a bulky "bypass" prosthesis. This stump revision enabled the patient to attain independent functional prosthetic ambulation. Although there is evidence of some recurrence of bone spur formation, this remains limited and asymptomatic.

Dysphagia associated with cricopharyngeal dysfunction.

In protracted forms of dysphagia associated with neuromuscular dysfunction, myotomy of the upper esophageal sphincter has been suggested. The literature, however, is unclear about the indications and outcome of this procedure. In this article, 3 cases are presented of dysphagia associated with the failure of relaxation of the cricopharyngeus during swallowing. Two patients had brain stem infarctions and the 3rd had an inflammatory disease of the brain stem. In all patients, barium swallow revealed aspiration of contrast material into the trachea with failure of relaxation of the cricopharyngeal sphincter. Indirect laryngoscopy demonstrated partial paralysis of one or both vocal cords. In one, an emg of the laryngeal muscles showed normal results. A complete evaluation by the speech pathologist failed to reveal abnormality of the oral musculature in all patients. All patients required gastrostomies for their nutritional needs. Therapeutic modification and control of rate, quantity and consistency of food along with counseling for prevention of aspiration. Oral feeding, without aspiration, was achieved in the 3 patients within the treatment period, allowing removal of the gastrostomy. Cricopharyngeal myotomy, therefore, was not necessary in these patients, despite the long duration of the swallowing problem.

Conditioning program for amputees with significant heart disease.

A program has been developed for training amputees with significant heart disease. In seven patients, a stress test was performed to evaluate cardiac functional capacity and heart rate response, utilizing both invasive and noninvasive techniques. Following this test, a conditioning program was instituted to improve cardiovascular function. At discharge and six week follow-up, all patients were able to ambulate at least 90 feet (27.4 meters) without excessive pulse rise or cardiac symptoms. This study demonstrates that by proper application of the principles of exercise physiology and cardiac pathophysiology, one should not necessarily deny a disabled patient wiith significant heart disease the opportunity to participate in a rehabilitation program.

Myocardial performance of disabled patients in a rehabilitation program.

Thirty-two patients with heart disease in addition to another disability were monitored continuously with the ten-hour electrocardiographic (ECG) tape. Eighteen of the 32 patients manifested abnormalities of rate, rhythm or ST-segment depression. Patients with arrhythmia eventually did better than those with other ECG abnormalities because drug therapy was successful in abolishing the abnormal rhythm in most cases. Those patients with excessive tachycardia did poorly as a group in so far as their rehabilitation program was concerned. This technique of continuous ECG monitoring may be helpful in assessing myocardial performance in these patients and in defining the prescription for rehabilitation therapy.