Clinical evaluation of a prosthetic suspension system: Looped silicone liner.

OBJECTIVES: This study aimed to evaluate and compare a newly designed suspension system with a common suspension in the market. STUDY DESIGN: Prospective study. METHODS: Looped liners with hook fastener and Iceross Dermo Liner with pin/lock system were mechanically tested using a tensile testing machine in terms of system safety. A total of 10 transtibial amputees participated in this study and were asked to use these two different suspension systems. The pistoning was measured between the liner and socket through a photographic method. Three static axial loading conditions were implemented, namely, 30, 60, and 90 N. Furthermore, subjective feedback was obtained. RESULTS: Tensile test results showed that both systems could safely tolerate the load applied to the prosthesis during ambulation. Clinical evaluation confirmed extremely low pistoning in both systems (i.e. less than 0.4 cm after adding 90 N traction load to the prosthesis). Subjective feedback also showed satisfaction with both systems. However, less traction at the end of the residual limb was reported while looped liner was used. CONCLUSION: The looped liner with hook fastener is safe and a good alternative for individuals with transtibial amputation as this system could solve some problems with the current systems. Clinical relevance The looped liner and hook fastener were shown to be good alternative suspension for people with lower limb amputation especially those who have difficulty to use and align the pin/lock systems. This system could safely tolerate centrifugal forces applied to the prosthesis during normal and fast walking.

Prosthesis donning and doffing questionnaire: Development and validation.

OBJECTIVES: To develop a questionnaire that specifically evaluates the ability of trans-tibial amputees to don and doff a prosthesis and to investigate the psychometric properties of the newly developed questionnaire. BACKGROUND: Prosthesis should be donned and doffed few times during the day and night; thus, it is important to measure ease of donning and doffing. STUDY DESIGN: A cross-sectional study. METHODS: The questionnaire was designed and evaluated by a group of experts. The final questionnaire was administered to 50 individuals with trans-tibial amputation. A test-retest study was also conducted on 20 amputees to assess the repeatability of questionnaire items. RESULTS: The prosthesis donning and doffing questionnaire was developed and tested through a pilot study. Based on Kappa index, the questionnaire items showed correlation coefficients greater than 0.7, which indicate good reliability and repeatability. The majority of the participants had good hand dexterity (80%) and could perform all types of grasps. The mean satisfaction scores with donning and doffing were 69.9 and 81.4, respectively. Most of the respondents needed to don and doff the prosthesis 3.44 times per day. Based on a 7-point score, the total scores ranged between 3 and 7. CONCLUSION: The prosthesis donning and doffing questionnaire items showed good psychometric properties. A scoring method was suggested based on the pilot sample, which requires further evaluation to be able to differentiate between more suspension types. A larger international multicenter evaluation is required in the future to measure the responsiveness of the scales. This questionnaire will be useful in the evaluation of the ability of amputees to don and doff a trans-tibial limb prosthesis. Clinical relevance Donning and doffing of prostheses are challenging tasks for many lower limb amputees. The prosthesis donning and doffing questionnaire, on its own or combined with other prosthetic evaluation questionnaires, has the potential to help manufacturers, clinicians, and researchers gain knowledge and improve the donning and doffing qualities of prostheses.

Assessment of tumourigenic potential in long-term cryopreserved human adipose-derived stem cells.

Cryopreservation represents an efficient way to preserve human mesenchymal stem cells (hMSCs) at early culture/passage, and allows pooling of cells to achieve sufficient cells required for off-the-shelf use in clinical applications, e.g. cell-based therapies and regenerative medicine. To fully apply cryopreserved hMSCs in a clinical setting, it is necessary to evaluate their biosafety, e.g. chromosomal abnormality and tumourigenic potential. To date, many studies have demonstrated that cryopreserved hMSCs display no chromosomal abnormalities. However, the tumourigenic potential of cryopreserved hMSCs has not yet been evaluated. In the present study, we cryopreserved human adipose-derived mesenchymal stem cells (hASCs) for 3 months, using a slow freezing method with various cryoprotective agents (CPAs), followed by assessment of the tumourigenic potential of the cryopreserved hASCs after thawing and subculture. We found that long-term cryopreserved hASCs maintained normal levels of the tumour suppressor markers p53, p21, p16 and pRb, hTERT, telomerase activity and telomere length. Further, we did not observe significant DNA damage or signs of p53 mutation in cryopreserved hASCs. Our findings suggest that long-term cryopreserved hASCs are at low risk of tumourigenesis. These findings aid in establishing the biosafety profile of cryopreserved hASCs, and thus establishing low hazardous risk perception with the use of long-term cryopreserved hASCs for future clinical applications. Copyright © 2016 John Wiley & Sons, Ltd.

Paper-based point-of-care testing for diagnosis of dengue infections.

Dengue endemic is a serious healthcare concern in tropical and subtropical countries. Although well-established laboratory tests can provide early diagnosis of acute dengue infections, access to these tests is limited in developing countries, presenting an urgent need to develop simple, rapid, and robust diagnostic tools. Point-of-care (POC) devices, particularly paper-based POC devices, are typically rapid, cost-effective and user-friendly, and they can be used as diagnostic tools for the prompt diagnosis of dengue at POC settings. Here, we review the importance of rapid dengue diagnosis, current dengue diagnostic methods, and the development of paper-based POC devices for diagnosis of dengue infections at the POC.

Paracrine Effects of Adipose-Derived Stem Cells on Matrix Stiffness-Induced Cardiac Myofibroblast Differentiation via Angiotensin II Type 1 Receptor and Smad7.

Human mesenchymal stem cells (hMSCs) hold great promise in cardiac fibrosis therapy, due to their potential ability of inhibiting cardiac myofibroblast differentiation (a hallmark of cardiac fibrosis). However, the mechanism involved in their effects remains elusive. To explore this, it is necessary to develop an in vitro cardiac fibrosis model that incorporates pore size and native tissue-mimicking matrix stiffness, which may regulate cardiac myofibroblast differentiation. In the present study, collagen coated polyacrylamide hydrogel substrates were fabricated, in which the pore size was adjusted without altering the matrix stiffness. Stiffness is shown to regulate cardiac myofibroblast differentiation independently of pore size. Substrate at a stiffness of 30 kPa, which mimics the stiffness of native fibrotic cardiac tissue, was found to induce cardiac myofibroblast differentiation to create in vitro cardiac fibrosis model. Conditioned medium of hMSCs was applied to the model to determine its role and inhibitory mechanism on cardiac myofibroblast differentiation. It was found that hMSCs secrete hepatocyte growth factor (HGF) to inhibit cardiac myofibroblast differentiation via downregulation of angiotensin II type 1 receptor (AT1R) and upregulation of Smad7. These findings would aid in establishment of the therapeutic use of hMSCs in cardiac fibrosis therapy in future.

An integrated lateral flow assay for effective DNA amplification and detection at the point of care.

Lateral flow assays (LFAs) have been extensively explored in nucleic acid testing (NAT) for medical diagnostics, food safety analysis and environmental monitoring. However, the amount of target nucleic acid in a raw sample is usually too low to be directly detected by LFAs, necessitating the process of amplification. Even though cost-effective paper-based amplification techniques have been introduced, they have always been separately performed from LFAs, hence increasing the risk of reagent loss and cross-contaminations. To date, integrating paper-based nucleic acid amplification into colorimetric LFA in a simple, portable and cost-effective manner has not been introduced. Herein, we developed an integrated LFA with the aid of a specially designed handheld battery-powered system for effective amplification and detection of targets in resource-poor settings. Interestingly, using the integrated paper-based loop-mediated isothermal amplification (LAMP)-LFA, we successfully performed highly sensitive and specific target detection, achieving a detection limit of as low as 3 × 10(3) copies of target DNA, which is comparable to the conventional tube-based LAMP-LFA in an unintegrated format. The device may serve in conjunction with a simple paper-based sample preparation to create a fully integrated paper-based sample-to-answer diagnostic device for point-of-care testing (POCT) in the near future.

The effects of hypoxia and serum-free conditions on the stemness properties of human adipose-derived stem cells.

The need to have a better and safer culture condition for expansion of human mesenchymal stem cells (MSCs) is crucial particularly to prevent infection and immune rejection. This is normally associated with the use of animal-based serum in the culture media for cell expansion. The aim of this study is to investigate alternative culture conditions which may provide better and safer environment for cell growth. In the present study, human adipose-derived stem cells (ASCs) at passage 3 were subjected to treatment in 4 conditions: (1) 21 % O2 with fetal bovine serum (FBS), (2) 21 % O2 without FBS, (3) 2 % O2 with FBS and (4) 2 % O2 without FBS followed by subsequent analysis of their phenotype, viability and functionality. We observed that ASCs cultured in all conditions present no significant phenotypic changes. It was found that ASCs cultured in 2 % O2 without serum showed an increase in viability and growth to a certain extent when compared to those cultured in 21 % O2 without serum. However, ASCs cultured in 2 % O2 without serum displayed a relatively low adipogenic and osteogenic potential. On the other hand, interestingly, there was a positive enhancement in chondrogenic differentiation of ASCs cultured in 21 % O2 without serum. Our findings suggest that different culture conditions may be suitable for different indications. In summary, ASCs cultured in serum-free condition can still survive, proliferate and undergo subsequent adipogenic, osteogenic and chondrogenic differentiation. Therefore, FBS is feasible to be excluded for culture of ASCs, which avoids clinical complications.

An integrated paper-based sample-to-answer biosensor for nucleic acid testing at the point of care.

With advances in point-of-care testing (POCT), lateral flow assays (LFAs) have been explored for nucleic acid detection. However, biological samples generally contain complex compositions and low amounts of target nucleic acids, and currently require laborious off-chip nucleic acid extraction and amplification processes (e.g., tube-based extraction and polymerase chain reaction (PCR)) prior to detection. To the best of our knowledge, even though the integration of DNA extraction and amplification into a paper-based biosensor has been reported, a combination of LFA with the aforementioned steps for simple colorimetric readout has not yet been demonstrated. Here, we demonstrate for the first time an integrated paper-based biosensor incorporating nucleic acid extraction, amplification and visual detection or quantification using a smartphone. A handheld battery-powered heating device was specially developed for nucleic acid amplification in POC settings, which is coupled with this simple assay for rapid target detection. The biosensor can successfully detect Escherichia coli (as a model analyte) in spiked drinking water, milk, blood, and spinach with a detection limit of as low as 10-1000 CFU mL(-1), and Streptococcus pneumonia in clinical blood samples, highlighting its potential use in medical diagnostics, food safety analysis and environmental monitoring. As compared to the lengthy conventional assay, which requires more than 5 hours for the entire sample-to-answer process, it takes about 1 hour for our integrated biosensor. The integrated biosensor holds great potential for detection of various target analytes for wide applications in the near future.

Sensitive biomolecule detection in lateral flow assay with a portable temperature-humidity control device.

Lateral flow assays (LFAs) have currently attracted broad interest for point-of-care (POC) diagnostics, but their application has been restricted by poor quantification and limited sensitivity. While the former has been currently solved to some extent by the development of handheld or smartphone-based readers, the latter has not been addressed fully, particularly the potential influences of environmental conditions (e.g., temperature and relative humidity (RH)), which have not yet received serious attention. The present study reports the use of a portable temperature-humidity control device to provide an optimum environmental requirement for sensitivity improvement in LFAs, followed by quantification by using a smartphone. We found that a RH beyond 60% with temperatures of 55-60°C and 37-40°C produced optimum nucleic acid hybridization and antigen-antibody interaction in LFAs, respectively representing a 10-fold and 3-fold signal enhancement over ambient conditions (25°C, 60% RH). We envision that in the future the portable device could be coupled with a fully integrated paper-based sample-to-answer biosensor for sensitive detection of various target analytes in POC settings.

Cryopreservation of Human Mesenchymal Stem Cells for Clinical Applications: Current Methods and Challenges.

Mesenchymal stem cells (MSCs) hold many advantages over embryonic stem cells (ESCs) and other somatic cells in clinical applications. MSCs are multipotent cells with strong immunosuppressive properties. They can be harvested from various locations in the human body (e.g., bone marrow and adipose tissues). Cryopreservation represents an efficient method for the preservation and pooling of MSCs, to obtain the cell counts required for clinical applications, such as cell-based therapies and regenerative medicine. Upon cryopreservation, it is important to preserve MSCs functional properties including immunomodulatory properties and multilineage differentiation ability. Further, a biosafety evaluation of cryopreserved MSCs is essential prior to their clinical applications. However, the existing cryopreservation methods for MSCs are associated with notable limitations, leading to a need for new or improved methods to be established for a more efficient application of cryopreserved MSCs in stem cell-based therapies. We review the important parameters for cryopreservation of MSCs and the existing cryopreservation methods for MSCs. Further, we also discuss the challenges to be addressed in order to preserve MSCs effectively for clinical applications.

Paper-based sample-to-answer molecular diagnostic platform for point-of-care diagnostics.

Nucleic acid testing (NAT), as a molecular diagnostic technique, including nucleic acid extraction, amplification and detection, plays a fundamental role in medical diagnosis for timely medical treatment. However, current NAT technologies require relatively high-end instrumentation, skilled personnel, and are time-consuming. These drawbacks mean conventional NAT becomes impractical in many resource-limited disease-endemic settings, leading to an urgent need to develop a fast and portable NAT diagnostic tool. Paper-based devices are typically robust, cost-effective and user-friendly, holding a great potential for NAT at the point of care. In view of the escalating demand for the low cost diagnostic devices, we highlight the beneficial use of paper as a platform for NAT, the current state of its development, and the existing challenges preventing its widespread use. We suggest a strategy involving integrating all three steps of NAT into one single paper-based sample-to-answer diagnostic device for rapid medical diagnostics in the near future.

Benchtop technologies for circulating tumor cells separation based on biophysical properties.

Circulating tumor cells (CTCs) are tumor cells that have detached from primary tumor site and are transported via the circulation system. The importance of CTCs as prognostic biomarker is leveraged when multiple studies found that patient with cutoff of 5 CTCs per 7.5 mL blood has poor survival rate. Despite its clinical relevance, the isolation and characterization of CTCs can be quite challenging due to their large morphological variability and the rare presence of CTCs within the blood. Numerous methods have been employed and discussed in the literature for CTCs separation. In this paper, we will focus on label free CTCs isolation methods, in which the biophysical and biomechanical properties of cells (e.g., size, deformability, and electricity) are exploited for CTCs detection. To assess the present state of various isolation methods, key performance metrics such as capture efficiency, cell viability, and throughput will be reported. Finally, we discuss the challenges and future perspectives of CTC isolation technologies.

Evaluation of postural steadiness in below-knee amputees when wearing different prosthetic feet during various sensory conditions using the Biodex® Stability System.

In recent years, computerized posturography has become an essential tool in quantitative assessment of postural steadiness in the clinical settings. The purpose of this study was to explore the ability of the Biodex(®) Stability System (BSS) to quantify postural steadiness in below-knee amputees. A convenience sample of 10 below-knee amputees participated in the study. The overall (OSI), anterior-posterior (APSI) and medial-lateral (MLSI) stability indexes as well as the percentage of time spent in left and right quadrants and four concentric zones were measured under altered sensory conditions while standing with solid ankle cushion heel (SACH), single-axis (SA) and energy storage and release (ESAR) feet. Significant difference was found between sensory conditions in SACH and ESAR feet for OSI (SACH, p = 0.002; ESAR, p = 0.005), APSI (SACH, p = 0.036; ESAR, p = 0.003) and MLSI (SACH, p = 0.008; ESAR, p = 0.05) stability indexes. The percentage of time spent in Zone A (0°-5°) was significantly greater than the other three concentric zones (p < 0.01). The loading time percentage on their intact limb (80%-94%) was significantly longer than the amputated limb (20%-6%) in all conditions for all three prosthetic feet. Below-knee amputees showed compromised postural steadiness when visual, proprioceptive or vestibular sensory input was altered. The findings highlight that the characteristics of postural stability in amputees can be clinically assessed by utilizing the outcomes produced by the BSS.

Phenotypic and functional characterization of long-term cryopreserved human adipose-derived stem cells.

Cryopreservation represents an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs) and allows pooling of cells via long-term storage for clinical applications, e.g., cell-based therapies. It is crucial to reduce freezing injury during the cryopreservation process by loading the ASCs with the optimum concentration of suitable cryoprotective agents (CPAs). In this study, human ASCs were preserved for 3 months in different combinations of CPAs, including 1) 0.25 M trehalose; 2) 5% dimethylsulfoxide (DMSO); 3) 10% DMSO; 4) 5% DMSO + 20% fetal bovine serum (FBS); 5) 10% DMSO + 20% FBS; 6) 10% DMSO + 90% FBS. Interestingly, even with a reduction of DMSO to 5% and without FBS, cryopreserved ASCs maintained high cell viability comparable with standard cryomedium (10% DMSO + 90% FBS), with normal cell phenotype and proliferation rate. Cryopreserved ASCs also maintained their differentiation capability (e.g., to adipocytes, osteocytes and chondrocytes) and showed an enhanced expression level of stemness markers (e.g., NANOG, OCT-4, SOX-2 and REX-1). Our findings suggest that 5% DMSO without FBS may be an ideal CPA for an efficient long-term cryopreservation of human ASCs. These results aid in establishing standardized xeno-free long-term cryopreservation of human ASCs for clinical applications.

In situ normoxia enhances survival and proliferation rate of human adipose tissue-derived stromal cells without increasing the risk of tumourigenesis.

Adipose tissue-derived stromal cells (ASCs) natively reside in a relatively low-oxygen tension (i.e., hypoxic) microenvironment in human body. Low oxygen tension (i.e., in situ normoxia), has been known to enhance the growth and survival rate of ASCs, which, however, may lead to the risk of tumourigenesis. Here, we investigated the tumourigenic potential of ASCs under their physiological condition to ensure their safe use in regenerative therapy. Human ASCs isolated from subcutaneous fat were cultured in atmospheric O2 concentration (21% O2) or in situ normoxia (2% O2). We found that ASCs retained their surface markers, tri-lineage differentiation potential, and self-renewal properties under in situ normoxia without altering their morphology. In situ normoxia displayed a higher proliferation and viability of ASCs with less DNA damage as compared to atmospheric O2 concentration. Moreover, low oxygen tension significantly up-regulated VEGF and bFGF mRNA expression and protein secretion while reducing the expression level of tumour suppressor genes p16, p21, p53, and pRb. However, there were no significant differences in ASCs telomere length and their relative telomerase activity when cultured at different oxygen concentrations. Collectively, even with high proliferation and survival rate, ASCs have a low tendency of developing tumour under in situ normoxia. These results suggest 2% O2 as an ideal culture condition for expanding ASCs efficiently while maintaining their characteristics.

Interface stress in socket/residual limb with transtibial prosthetic suspension systems during locomotion on slopes and stairs.

OBJECTIVE: This study aimed to compare the effects of different suspension methods on the interface stress inside the prosthetic sockets of transtibial amputees when negotiating ramps and stairs. DESIGN: Three transtibial prostheses, with a pin/lock system, a Seal-In system, and a magnetic suspension system, were created for the participants in a prospective study. Interface stress was measured as the peak pressure by using the F-socket transducers during stairs and ramp negotiation. RESULTS: Twelve individuals with transtibial amputation managed to complete the experiments. During the stair ascent and descent, the greatest peak pressure was observed in the prosthesis with the Seal-In system. The magnetic prosthetic suspension system caused significantly different peak pressure at the anterior proximal region compared with the pin/lock (P = 0.022) and Seal-In (P = 0.001) during the stair ascent. It was also observed during the stair descent and ramp negotiation. CONCLUSIONS: The prostheses exhibited varying pressure profiles during the stair and ramp ascent. The prostheses with the pin/lock and magnetic suspension systems exhibited lower peak pressures compared with the Seal-In system. The intrasystem pressure distribution at the anterior and posterior regions of the residual limb was fairly homogenous during the stair and ramp ascent and descent. Nevertheless, the intrasystem pressure mapping revealed a significant difference among the suspension types, particularly at the anterior and posterior sensor sites.

Endothelial cell responses in terms of adhesion, proliferation, and morphology to stiffness of polydimethylsiloxane elastomer substrates.

Extracellular environments can regulate cell behavior because cells can actively sense their mechanical environments. This study evaluated the adhesion, proliferation and morphology of endothelial cells on polydimethylsiloxane (PDMS)/alumina (Al2 O3 ) composites and pure PDMS. The substrates were prepared from pure PDMS and its composites with 2.5, 5, 7.5, and 10 wt % Al2 O3 at a curing temperature of 50°C for 4 h. The substrates were then characterized by mechanical, structural, and morphological analyses. The cell adhesion, proliferation, and morphology of cultured bovine aortic endothelial (BAEC) cells on substrate materials were evaluated by using resazurin assay and 1,1'-dioctadecyl-1,3,3,3',3'-tetramethylindocarbocyanine perchlorate-acetylated LDL (Dil-Ac-LDL) cell staining, respectively. The composites (PDMS/2.5, 5, 7.5, and 10 wt % Al2 O3 ) exhibited higher stiffness than the pure PDMS substrate. The results also revealed that stiffer substrates promoted endothelial cell adhesion and proliferation and also induced spread morphology in the endothelial cells compared with lesser stiff substrates. Statistical analysis showed that the effect of time on cell proliferation depended on stiffness. Therefore, this study concludes that the addition of different Al2 O3 percentages to PDMS elevated substrate stiffness which in turn increased endothelial cell adhesion and proliferation significantly and induced spindle shape morphology in endothelial cells.

Comparative study between Dermo, Pelite, and Seal-In X5 liners: effect on patient's satisfaction and perceived problems.

PURPOSE: This study aimed to compare the effect of satisfaction and perceived problems between Pelite, Dermo with shuttle lock, and Seal-In X5 liners on the transtibial amputees. MATERIAL AND METHODS: A total of thirty transtibial amputees (17 male, 13 female) volunteered to take part in this research. Two prostheses were fabricated for each participant. Prosthetic Evaluation Questionnaire (PEQ) was filled in by the participants with the three liners. RESULTS: The statistics highlight that Dermo liner showed significantly higher score (P = 0.05) in walking, walking on uneven surfaces, stairs walking, fitting, donning/doffing, sitting, suspension, and overall satisfaction with Dermo liner compared with Seal-In X5 and Pelite liners. Overall satisfaction was 34% higher with Dermo liner than Seal-In X5 liner and 28% higher than Pelite liner. Participants reported less problems with Dermo liner and significant differences (P < 0.05) were recorded between the three liners in sweating, skin irritation, frustration, and pain compared with Seal-In X5 and Pelite liners. CONCLUSION: Participants experienced high level of satisfaction and practiced fewer problems with Dermo liner. These results showed that there is good indication to believe that Dermo liner might be a good choice for transtibial users and might help the clinicians and prosthetic practitioners in selection criteria of prosthetic liners.