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.
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.
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.
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.
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.
Adipose-derived stem cells (ASCs) have been found adapted to a specific niche with low oxygen tension (hypoxia) in the body. As an important component of this niche, oxygen tension has been known to play a critical role in the maintenance of stem cell characteristics. However, the effect of O2 tension on their functional properties has not been well determined. In this study, we investigated the effects of O2 tension on ASCs stemness, differentiation and proliferation ability. Human ASCs were cultured under normoxia (21% O2) and hypoxia (2% O2). We found that hypoxia increased ASC stemness marker expression and proliferation rate without altering their morphology and surface markers. Low oxygen tension further enhances the chondrogenic differentiation ability, but reduces both adipogenic and osteogenic differentiation potential. These results might be correlated with the increased expression of HIF-1α under hypoxia. Taken together, we suggest that growing ASCs under 2% O2 tension may be important in expanding ASCs effectively while maintaining their functional properties for clinical therapy, particularly for the treatment of cartilage defects.
Poor suspension increases slippage of the residual limb inside the socket during ambulation. The main purpose of this article is to evaluate the pistoning at the prosthetic liner-socket interface during gait and assess patients' satisfaction with two different liners. Two prostheses with seal-in and locking liners were fabricated for each of the 10 subjects with transtibial amputation. The Vicon motion system was used to measure the pistoning during gait. The subjects were also asked to complete a Prosthesis Evaluation Questionnaire. The results revealed higher pistoning inside the socket during gait with the locking liner than with the seal-in liner (p < 0.05). The overall satisfaction with the locking liner was higher (p < 0.05) because of the relative ease with which the patients could don and doff the device. As such, pistoning may not be the main factor that determines patients' overall satisfaction with the prosthesis and other factors may also contribute to comfort and satisfaction with prostheses. The article also verifies the feasibility of the Vicon motion system for measuring pistoning during gait.
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.
Knee osteoarthritis is a common cause of disability which influences the quality of life. It is associated with impaired knee joint proprioception, which affects postural stability. Postural stability is critical for mobility and physical activities. Different types of treatment including nonsurgical and surgical are used for knee osteoarthritis. Hyaluronic acid injection is a nonsurgical popular treatment used worldwide. The aim of this study was to demonstrate the effect of hyaluronic acid injections on postural stability in individuals with bilateral knee osteoarthritis. Fifty patients aged between 50 and 70 years with mild and moderate bilateral knee osteoarthritis participated in our study. They were categorized into treatment (n = 25) and control (n = 25) groups. The treatment group received five weekly hyaluronic acid injections for both knees, whereas the control group did not receive any treatment. Postural stability and fall risk were assessed using the Biodex Stability System and clinical "Timed Up and Go" test. All the participants completed the study. The treatment group showed significant decrease in postural stability and fall risk scores after five hyaluronic acid injections. In contrast, the control group showed significant increase. This study illustrated that five intra-articular hyaluronic acid injections could significantly improve postural stability and fall risk in bilateral knee osteoarthritis patients. This trial is registered with: NCT02063373.
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.
The suspension system and socket fitting of artificial limbs have major roles and vital effects on the comfort, mobility, and satisfaction of amputees. This paper introduces a new pneumatic suspension system that overcomes the drawbacks of current suspension systems in donning and doffing, change in volume during daily activities, and pressure distribution in the socket-stump interface. An air pneumatic suspension system (APSS) for total-contact sockets was designed and developed. Pistoning and pressure distribution in the socket-stump interface were tested for the new APSS. More than 95% of the area between each prosthetic socket and liner was measured using a Tekscan F-Scan pressure measurement which has developed matrix-based pressure sensing systems. The variance in pressure around the stump was 8.76 kPa. APSS exhibits less pressure concentration around the stump, improved pressure distribution, easy donning and doffing, adjustability to remain fitted to the socket during daily activities, and more adaptability to the changes in stump volume. The volume changes were adjusted by utility of air pressure sensor. The vertical displacement point and reliability of suspension were assessed using a photographic method. The optimum pressure in every level of loading weight was 55 kPa, and the maximum displacement was 6 mm when 90 N of weight was loaded.
This study aimed to evaluate the effects of prosthetic foot types on the postural stability among transtibial amputees when standing on different support surfaces.
Socket is an important part of every prosthetic limb as an interface between the residual limb and prosthetic components. Biomechanics of socket-residual limb interface, especially the pressure and force distribution, have effect on patient satisfaction and function. This paper aimed to review and evaluate studies conducted in the last decades on the design of socket, in-socket interface pressure measurement, and socket biomechanics. Literature was searched to find related keywords with transtibial amputation, socket-residual limb interface, socket measurement, socket design, modeling, computational modeling, and suspension system. In accordance with the selection criteria, 19 articles were selected for further analysis. It was revealed that pressure and stress have been studied in the last decaeds, but quantitative evaluations remain inapplicable in clinical settings. This study also illustrates prevailing systems, which may facilitate improvements in socket design for improved quality of life for individuals ambulating with transtibial prosthesis. It is hoped that the review will better facilitate the understanding and determine the clinical relevance of quantitative evaluations.
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.
Balance is essential for mobility and performing activities of daily living. People with knee osteoarthritis display impairment in knee joint proprioception. Thus, the aim of this study was to evaluate balance and risk of fall in individuals with bilateral mild and moderate knee osteoarthritis. Sixty subjects aged between 50 and 70 years volunteered in this study. They were categorized into three groups which were healthy (n = 20), mild (n = 20) and moderate (n = 20) bilateral knee osteoarthritis groups. Dynamic and static balance and risk of fall were assessed using Biodex Stability System. In addition, Timed Up and Go test was used as a clinical test for balance. Results of this study illustrated that there were significant differences in balance (dynamic and static) and risk of fall between three groups. In addition, the main (most significant) difference was found to be between healthy group and moderate group. Furthermore, on clinical scoring of balance, the "Timed Up and Go" test, all three groups showed significant difference. In conclusion, bilateral knee osteoarthritis impaired the balance and increased the risk of fall, particularly in people with moderate knee osteoarthritis.
Prosthetic suspension system is an important component of lower limb prostheses. Suspension efficiency can be best evaluated during one of the vital activities of daily living, i.e. walking. A new magnetic prosthetic suspension system has been developed, but its effects on gait biomechanics have not been studied. This study aimed to explore the effect of suspension type on kinetic and kinematic gait parameters during level walking with the new suspension system as well as two other commonly used systems (the Seal-In and pin/lock). Thirteen persons with transtibial amputation participated in this study. A Vicon motion system (six cameras, two force platforms) was utilized to obtain gait kinetic and kinematic variables, as well as pistoning within the prosthetic socket. The gait deviation index was also calculated based on the kinematic data. The findings indicated significant difference in the pistoning values among the three suspension systems. The Seal-In system resulted in the least pistoning compared with the other two systems. Several kinetic and kinematic variables were also affected by the suspension type. The ground reaction force data showed that lower load was applied to the limb joints with the magnetic suspension system compared with the pin/lock suspension. The gait deviation index showed significant deviation from the normal with all the systems, but the systems did not differ significantly. Main significant effects of the suspension type were seen in the GRF (vertical and fore-aft), knee and ankle angles. The new magnetic suspension system showed comparable effects in the remaining kinetic and kinematic gait parameters to the other studied systems. This study may have implications on the selection of suspension systems for transtibial prostheses. Trial registration: Iranian Registry of Clinical Trials IRCT2013061813706N1.
Numerous protocols for the isolation of bovine aortic endothelial cells have been described in the previous literature. However, these protocols prevent researchers from obtaining the pure population of endothelial cells. Thus, this study aimed to develop a new and economical method for the isolation of pure endothelial cells by introducing a new strategy to the enzymatic digestion method proposed by previous researchers.
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.
The knee adduction moment represents the medial knee joint load, and greater value is associated with higher load. In people with knee osteoarthritis, it is important to apply proper treatment with the least side effects to reduce knee adduction moment and, consequently, reduce medial knee joint load. This reduction may slow the progression of knee osteoarthritis. The research team performed a literature search of electronic databases. The search keywords were as follows: knee osteoarthritis, knee adduction moment, exercise program, exercise therapy, gait retraining, gait modification and knee joint loading. In total, 12 studies were selected, according to the selection criteria. Findings from previous studies illustrated that exercise and gait retraining programs could alter knee adduction moment in people with knee osteoarthritis. These treatments are noninvasive and nonpharmacological which so far have no or few side effects, as well as being low cost. The results of this review revealed that gait retraining programs were helpful in reducing the knee adduction moment. In contrast, not all the exercise programs were beneficial in reducing knee adduction moment. Future studies are needed to indicate best clinical exercise and gait retraining programs, which are most effective in reducing knee adduction moment in people with knee osteoarthritis.
Study design: systematic review
Good prosthetic suspension system secures the residual limb inside the prosthetic socket and enables easy donning and doffing. This study aimed to introduce, evaluate and compare a newly designed prosthetic suspension system (HOLO) with the current suspension systems (suction, pin/lock and magnetic systems).