The degradation and performance of electrospun supramolecular vascular scaffolds examined upon in vitro enzymatic exposure.

To maintain functionality during in situ vascular regeneration, the rate of implant degradation should be closely balanced by neo-tissue formation. It is unknown, however, how the implant's functionality is affected by the degradation of the polymers it is composed of. We therefore examined the macro- and microscopic features as well as the mechanical performance of vascular scaffolds upon in vitro enzymatic degradation. Three candidate biomaterials with supramolecularly interacting bis-urea (BU) hard blocks ('slow-degrading' polycarbonate-BU (PC-BU), 'intermediate-degrading' polycarbonate-ester-BU (PC(e)-BU), and 'fast-degrading' polycaprolactone-ester-BU (PCL-BU)) were synthesized and electrospun into microporous scaffolds. These materials possess a sequence-controlled macromolecular structure, so their susceptibility to degradation is tunable by controlling the nature of the polymer backbone. The scaffolds were incubated in lipase and monitored for changes in physical, chemical, and mechanical properties. Remarkably, comparing PC-BU to PC(e)-BU, we observed that small changes in macromolecular structure led to significant differences in degradation kinetics. All three scaffold types degraded via surface erosion, which was accompanied by fiber swelling for PC-BU scaffolds, and some bulk degradation and a collapsing network for PCL-BU scaffolds. For the PC-BU and PC(e)-BU scaffolds this resulted in retention of mechanical properties, whereas for the PCL-BU scaffolds this resulted in stiffening. Our in vitro study demonstrates that vascular scaffolds, electrospun from sequence-controlled supramolecular materials with varying ester contents, not only display different susceptibilities to degradation, but also degrade via different mechanisms. STATEMENT OF SIGNIFICANCE: One of the key elements to successfully engineer vascular tissues in situ, is to balance the rate of implant degradation and neo-tissue formation. Due to their tunable properties, supramolecular polymers can be customized into attractive biomaterials for vascular tissue engineering. Here, we have exploited this tunability and prepared a set of polymers with different susceptibility to degradation. The polymers, which were electrospun into microporous scaffolds, displayed not only different susceptibilities to degradation, but also obeyed different degradation mechanisms. This study illustrates how the class of supramolecular polymers continues to represent a promising group of materials for tissue engineering approaches.

Hydrolytic and oxidative degradation of electrospun supramolecular biomaterials: In vitro degradation pathways.

The emerging field of in situ tissue engineering (TE) of load bearing tissues places high demands on the implanted scaffolds, as these scaffolds should provide mechanical stability immediately upon implantation. The new class of synthetic supramolecular biomaterial polymers, which contain non-covalent interactions between the polymer chains, thereby forming complex 3D structures by self assembly. Here, we have aimed to map the degradation characteristics of promising (supramolecular) materials, by using a combination of in vitro tests. The selected biomaterials were all polycaprolactones (PCLs), either conventional and unmodified PCL, or PCL with supramolecular hydrogen bonding moieties (either 2-ureido-[1H]-pyrimidin-4-one or bis-urea units) incorporated into the backbone. As these materials are elastomeric, they are suitable candidates for cardiovascular TE applications. Electrospun scaffold strips of these materials were incubated with solutions containing enzymes that catalyze hydrolysis, or solutions containing oxidative species. At several time points, chemical, morphological, and mechanical properties were investigated. It was demonstrated that conventional and supramolecular PCL-based polymers respond differently to enzyme-accelerated hydrolytic or oxidative degradation, depending on the morphological and chemical composition of the material. Conventional PCL is more prone to hydrolytic enzymatic degradation as compared to the investigated supramolecular materials, while, in contrast, the latter materials are more susceptible to oxidative degradation. Given the observed degradation pathways of the examined materials, we are able to tailor degradation characteristics by combining selected PCL backbones with additional supramolecular moieties. The presented combination of in vitro test methods can be employed to screen, limit, and select biomaterials for pre-clinical in vivo studies targeted to different clinical applications.

Fatigue in persons with subacute spinal cord injury who are dependent on a manual wheelchair.

STUDY DESIGN: Cross-sectional. OBJECTIVES: To determine the prevalence and severity of fatigue in persons with subacute spinal cord injury (SCI), assess whether demographic and lesion characteristics are related to fatigue and determine the relationship with physical fitness and physical behavior. SETTING: Measurements were performed 2 months before discharge from inpatient rehabilitation. METHODS: Thirty-six persons with subacute SCI, dependent on a manual wheelchair, mean age 43±15 and 83% men, completed the Fatigue Severity Scale (FSS). FSS scores >4 indicated fatigue. We recorded age and lesion characteristics, measured body mass index, measured peak power output and peak oxygen uptake during a maximal handcycling test and determined physical behavior using an accelerometer-based activity monitor. T-tests were used to test for differences in fatigue between subgroups based on age and lesion characteristics, and regression analyses to assess the relationship with physical fitness and physical behavior. RESULTS: Mean FSS was 3.3±1.3. Fatigue, including severe fatigue, was prevalent in 31% (95% confidence interval: 16-46) of participants compared with 18% in the general population. Furthermore, mean fatigue was significantly higher in persons with incomplete compared with complete lesions (t=2.22, P=0.03). Mean scores between other subgroups did not differ significantly. Of the physical fitness and physical behavior measures, only peak oxygen uptake tended to be related to more fatigue (B=-1.47, P=0.05). CONCLUSION: Fatigue was prevalent and is of concern in persons with subacute SCI. Those with incomplete lesions seem to be at higher risk. Because fatigue is known to persist among persons with SCI, interventions to reduce fatigue seem necessary.

Evaluation of strained alkynes for Cu-free click reaction in live mice.

INTRODUCTION: We report on our evaluation of the strain-promoted cyclooctyne-azide cycloaddition reaction for use in tumor pretargeting, comprising a side-by-side comparison of probes 1-3 bearing three distinct cyclooctyne moieties based respectively on the 1st and 2nd generation difluorinated cyclooctyne and the 1st generation dibenzocyclooctyne. METHODS: The probes were synthesized and labeled with (177)Lu with high yields. The probe stability and reactivity towards azides were evaluated in PBS and mouse serum, and their blood clearance, biodistribution and in vivo reactivity were evaluated in tumor-free mice. RESULTS: In serum the three probes exhibited sufficient stability for a pretargeting application with half-lives of 12-19h. In PBS, probes 2 and 3 were more reactive towards azido-conjugated Rituximab (Rtx-N3) than 1, but in contrast to 1, their reactivity decreased in mouse serum and mouse serum albumin solutions, as a result of covalent and non-covalent interactions with albumin. Biodistribution data confirmed the interactions with serum proteins in circulation: (177)Lu-1 showed a fast elimination from blood (t1/2,ß = 0.31h), while (177)Lu-2 and (177)Lu-3 were retained in blood for longer periods of time (t1/2,ß = 1.08 and 3.58h, respectively). Dual isotope biodistribution experiments assessing the reaction between (125)I-Rtx-N3 and (177)Lu-1-3 in circulation in mice showed a very limited retention of 2 and 3 in blood rich organs, indicating a minimal reactivity, while no such retention was observed for 1. CONCLUSION: The low reactivity of the studied cyclooctynes, and their serum interactions preclude their use at the low in vivo concentrations typical for pretargeting applications.

Long-term patient satisfaction after reconstructive upper extremity surgery to improve arm-hand function in tetraplegia.

STUDY DESIGN: There is a paucity of literature about satisfaction after reconstructive surgery to improve upper limb function in persons with tetraplegia. The present literature describes mainly functional outcomes. OBJECTIVES: To evaluate long-term satisfaction after reconstructive upper extremity surgery in persons with tetraplegia. SETTING: Two rehabilitation centers in the Netherlands. METHOD: A three-part questionnaire consisting questions regarding satisfaction, activities, occupation, changes in functional ability and willingness to undergo the surgeries again was used. Internal reliability of the questionnaire was verified by factor analysis and calculation of Cronbach's alpha. RESULTS: In total, 39 out of 55 persons (70.9%) participated in the study. The participants' responses to questions about satisfaction, activities and occupation were positive in 73.5, 67.6 and 35.0%, respectively. Nearly 81% improved their functional ability. Approximately 65% of the participants were willing to undergo elbow extension surgery again and 77.1% expressed their willingness to undergo hand/wrist surgery again. Significant positive correlation was found between willingness to have surgery again and improvement in activities and occupation: Spearman's correlation coefficients: activities-elbow extension 0.63 (P=0.003), activities-hand/wrist 0.57 (P<0.001), occupation-elbow extension 0.53 (P=0.025), occupation-hand/wrist 0.57 (P=0.001). Differences between the subgroups who would have surgery again and those who would refrain were also significant; one-way analysis of variance for activities (F=9.54, P<0.01) and for occupation (F=6.60, P<0.02). CONCLUSION: In the Netherlands, the majority of persons with tetraplegia who underwent reconstructive upper extremity surgery were satisfied with the results. This was related to improvement in activities and occupation.

Is manual wheelchair satisfaction related to active lifestyle and participation in people with a spinal cord injury?

STUDY DESIGN: Cross-sectional study. OBJECTIVES: To describe the satisfaction of the manual wheelchair user with hand rim wheelchair-related aspects (for example, dimensions, weight and comfort) and wheelchair service-related aspects and to determine the relationship between wheelchair users' satisfaction, personal and lesion characteristics, and active lifestyle and participation in persons with a spinal cord injury (SCI). SETTING: Eight Dutch rehabilitation centers with a specialized SCI unit. METHODS: The Dutch version of the Quebec user evaluation of satisfaction with assistive technology (D-QUEST) was filled out by 109 participants 1 year after discharge from inpatient SCI rehabilitation. Relationships between the D-QUEST scores and personal and lesion characteristics, and active lifestyle and participation (physical activity scale for individuals with physical disabilities (PASIPD), Utrecht activity list (UAL), mobility range and social behavior subscales of the SIP68 (SIPSOC)) were determined. RESULTS: A high level of satisfaction was found with wheelchair-related aspects. The participants were less satisfied with the service-related aspects. Participants with an incomplete lesion were slightly more satisfied regarding both aspects than those with a complete lesion. A higher satisfaction regarding wheelchair dimensions and a higher overall satisfaction were related to a more active lifestyle. Persons who were more satisfied with the simplicity of use of the wheelchair had a better participation score. CONCLUSION: Dutch persons with SCI are in general quite satisfied with their hand rim wheelchair. Some aspects of the wheelchair (dimensions and simplicity of use) are important to optimize as these are related to an active lifestyle and participation.

Assessment of upper extremity muscle function in persons with tetraplegia.

The objective was to investigate the actual level of muscle function impairment in tetraplegic persons and, inextricably related to this, the possibilities to compensate function loss using new muscle coalitions. In this cross-sectional group study, 20 persons with a cervical spinal cord injury (SCI) at segmental levels C5C6 or C7C8 and 10 control persons participated. Activity from 21 upper extremity and trunk muscles was recorded during standardised gross upper extremity task performance. No substantial differences in main activation patterns were found between C7C8 and control subjects. In contrast, main activation patterns in C5C6 persons showed an absence of selectivity, which may be explained by the participants activating every controllable muscle in an attempt to perform maximally. In order to identify more intricate differences in muscle activation between control and C7C8 persons a fine motor function task may be necessary. Muscle activation patterns during arm task performance were stable in all three groups.

Dendritic PARACEST contrast agents for magnetic resonance imaging.

MRI contrast agents based on chemical exchange-dependent saturation transfer (CEST), such as Yb(III)DOTAM complexes, are highly suitable for pH mapping. In this paper, the synthesis of Yb(III)DOTAM-functionalized poly(propylene imine) dendrimers is described. The applicability of these dendritic PARACEST MRI agents for pH mapping has been evaluated on a 7 T NMR spectrometer and on a 3 T clinical MRI scanner. As expected, based on the different numbers of exchangeable amide protons, the lowest detectable concentration of the first and third generation dendritic PARACEST agents is by a respective factor of about 4 and 16 lower than that of a mononuclear reference complex. The pH dependence of the CEST effect observed for these compounds depends on the generation of the poly(propylene imine) dendrimer. Upon going to higher generations of the Yb(III)DOTAM-terminated dendrimer, a shift of the maximum CEST effect towards lower pH values was observed. This allows for a fine-tuning of the responsive pH region by varying the dendritic framework.

Experienced and physiological fatigue in neuromuscular disorders.

OBJECTIVE: Fatigue has been described as a typical symptom of neurological diseases. It might be caused both by changes at the peripheral and at the central level. This study measured the level of experienced fatigue and physiological correlates of fatigue in three genetically defined neuromuscular disorders. METHODS: Sixty-five facioscapulohumeral dystrophy (FSHD), 79 classical myotonic dystrophy (DM), 73 hereditary motor and sensory neuropathy type I (HMSN) patients and 24 age-matched healthy controls made a 2-min sustained maximal voluntary contraction of the biceps brachii muscle. Experienced fatigue at the current moment was assessed with the abbreviated fatigue questionnaire just before the physiological measurement. Peripheral fatigue was quantified by comparing the amplitudes of an initial and a final stimulated force response during rest. Muscle fibre conduction velocity was determined from a 5-channel surface EMG recording in order to show peripheral changes during the contraction. Central aspects of fatigue were measured using superimposed electrical endplate stimulation. RESULTS: Patients showed an increased level of experienced fatigue. Total physiological and peripheral fatigue were smaller in patients compared to controls, and central fatigue was normal. The most interesting result of this study was the presence of a large central activation failure (CAF) in all groups of neuromuscular patients; they showed CAF values of 36-41% already directly at the start of sustained contraction, whereas the control group showed only 12%. CAF slightly correlated with the level of experienced fatigue just before the test. CONCLUSIONS: The cause of the large CAF in patients is unclear. Reduced concentration, motivation or effort can lead to lower central activation. In neuromuscular patients especially fear of physical activity or fear to damage the muscle or nerve tissue may contribute. Besides, also physiological feedback mechanisms or changes at the motocortical level may be a cause of reduced central activation. SIGNIFICANCE: For the clinician it is important to know that experienced fatigue is part of the clinical spectrum of neuromuscular patients. Besides, the weakness in these patients is aggravated by reduced central activation. Potentially, both problems could be subject of an intervention.

Effects of prosthesis alignment on pressure distribution at the stump/socket interface in transtibial amputees during unsupported stance and gait.

OBJECTIVE: To assess the effects of antero-posterior realignment of the prosthesis on pressure distribution at the stump/socket interface in unilateral transtibial amputees during unsupported stance and gait. DESIGN: Cross-sectional group study. SETTING: Outpatient rehabilitation clinic. SUBJECTS: Seventeen unilateral transtibial amputees. INTERVENTION: Antero-posterior prosthesis realignment using 0.5 cm heel or forefoot wedging. MEASURES: Pressure distribution changes at several sites at the stump/socket interface, expressed as peak pressure, peak pressure time, sustained submaximal load (MP80+) and time period of MP80+ (TP80+). RESULTS: During stance, heel wedging led to a 30.4% decrease in mean pressure near the patella tendon and a 40.0% increase near the distal tibia site, whereas forefoot wedging led to a 30.0% decrease in pressure in the tibial end region. Antero-posterior ankle realignment did not lead to significant changes near the fibular head. During gait only heel wedging led to a 11.5% increase in peak pressure in the tibia end region. Mean peak pressure time increased 22.7% using heel wedging and decreased 8.8% using forefoot wedging. MP80+ increased 23.8% in the tibia end region during heel wedging and 17.0% in the subpatellar region during forefoot wedging. CONCLUSIONS: Antero-posterior realignment of the ankle joint led to an inverse loading and unloading of the subpatellar region versus tibial end region. However, results indicate that stump/socket interface pressure during stance seems not to be highly predictive as to pressure behaviour during gait. Ankle joint realignment led to significant differences in temporal sequences of tissue (un-)loading in the subpatellar area during gait.

Ultrastructural localization of a chondroitinase-sensitive, cuprolinic blue-positive filament in developing mouse lung.

By use of the cationic dye Cuprolinic Blue in a critical electrolyte concentration method, the lungs of mice ranging from the late fetal stage (17 days of gestation) to the puberal stage (27 days) were surveyed for their proteoglycans. A large Cuprolinic Blue-positive filament is present within the connective tissue of lungs of late fetal and young postnatal mice. It is mostly located at the boundary between large extracellular matrix structures and electron microscopically empty areas, but sometimes also at the surface of fibroblast-like cells. The stainability of the filament disappears after treatment with chondroitinase ABC or chondroitinase AC, but not after treatment with nitrous acid. The Cuprolinic Blue-positive structure appears to be most abundant around 2 days postnatally. From day 10 on, its number decreases dramatically, and it can be no longer observed in the lungs of 27-day-old mice.

Isolation and characterization of a collagen fibril-associated dermatan sulphate proteoglycan from bovine lung.

Dermatan sulphate proteoglycans have been extracted from bovine lung with 2.0 M CaCl2 and isolated using CsCl density gradient centrifugation, DEAE ion-exchange chromatography, gel chromatography and preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Ultrastructurally these proteoglycans are specifically associated with collagen fibrils. Dermatan sulphate (Mr 15.10(3)-35.10(3), with a strong prevalence for the higher Mr) is link via an O-glycosidic bond to a protein core, which is rich in Asx, Glx and Leu. Of the total uronic acid, 91% is iduronic acid. A part of the glucuronic acid residues is located near the protein core and a large cluster of disaccharides is devoid of glucuronic acid residues. An inhibition enzyme immunoassay has been developed to quantitate the proteoglycan. A model for the interaction between dermatan sulphate proteoglycans and collagen fibrils is proposed.