Extracellular matrices have drawn attention in tissue engineering as potential biomaterials for scaffold fabrication because of their bioactive components. Noninvasive techniques of scaffold fabrication and cross-linking treatments are believed to maintain the integrity of bioactive molecules while providing proper architectural and mechanical properties. Cartilage matrix derived scaffolds are designed to support the maintenance of chondrocytes and provide proper signals for differentiation of chondroinducible cells. Chondroinductive potential of bovine articular cartilage matrix derived porous scaffolds on human dermal fibroblasts and the effect of scaffold shrinkage on chondrogenesis were investigated. An increase in sulfated glycosaminoglycans production along with upregulation of chondrogenic genes confirmed that physically treated cartilage matrix derived scaffolds have chondrogenic potential on human dermal fibroblasts.
The osteogenic potential of human adipose-derived stem cells (HADSCs) co-cultured with human osteoblasts (HOBs) using selected HADSCs/HOBs ratios of 1:1, 2:1, and 1:2, respectively, is evaluated. The HADSCs/HOBs were seeded on electrospun three-dimensional poly[(R)-3-hydroxybutyric acid] (PHB) blended with bovine-derived hydroxyapatite (BHA). Monocultures of HADSCs and HOBs were used as control groups. The effects of PHB-BHA scaffold on cell proliferation and cell morphology were assessed by AlamarBlue assay and field emission scanning electron microscopy. Cell differentiation, cell mineralization, and osteogenic-related gene expression of co-culture HADSCs/HOBs were examined by alkaline phosphatase (ALP) assay, alizarin Red S assay, and quantitative real time PCR, respectively. The results showed that co-culture of HADSCs/HOBs, 1:1 grown into PHB-BHA promoted better cell adhesion, displayed a significant higher cell proliferation, higher production of ALP, extracellular mineralization and osteogenic-related gene expression of run-related transcription factor, bone sialoprotein, osteopontin, and osteocalcin compared to other co-culture groups. This result also suggests that the use of electrospun PHB-BHA in a co-culture HADSCs/HOBs system may serve as promising approach to facilitate osteogenic differentiation activity of HADSCs through direct cell-to-cell contact with HOBs.
Articular cartilage is a tissue specifically adapted to a specific niche with a low oxygen tension (hypoxia), and the presence of such conditions is a key factor in regulating growth and survival of chondrocytes. Zinc deficiency has been linked to cartilage-related disease, and presence of Zinc is known to provide antibacterial benefits, which makes its inclusion attractive in an in vitro system to reduce infection. Inclusion of 1% zinc oxide nanoparticles (ZnONP) in poly octanediol citrate (POC) polymer cultured in hypoxia has not been well determined. In this study we investigated the effects of ZnONP on chondrocyte proliferation and matrix synthesis cultured under normoxia (21% O2 ) and hypoxia (5% O2 ). We report an upregulation of chondrocyte proliferation and sulfated glycosaminoglycan (S-GAG) in hypoxic culture. Results demonstrate a synergistic effect of oxygen concentration and 1% ZnONP in up-regulation of anabolic gene expression (Type II collagen and aggrecan), and a down regulation of catabolic (MMP-13) gene expression. Furthermore, production of transcription factor hypoxia-inducible factor 1A (HIF-1A) in response to hypoxic condition to regulate chondrocyte survival under hypoxia is not affected by the presence of 1% ZnONP. Presence of 1% ZnONP appears to act to preserve homeostasis of cartilage in its hypoxic environment.
Mechanical loading modulates cartilage homeostasis through the control of matrix synthesis and catabolism. However, the mechanotransduction pathways through which chondrocytes detect different loading conditions remain unclear. The present study investigated the influence of cyclic compression on intracellular Ca2+ signalling using the well-characterised chondrocyte-agarose model. Cells labelled with Fluo4 were visualised using confocal microscopy following a period of 10 cycles of compression between 0% and 10% strain. In unstrained agarose constructs, not subjected to cyclic compression, a subpopulation of approximately 45% of chondrocytes exhibited spontaneous global Ca2+ transients with mean transient rise and fall times of 19.4 and 29.4 sec, respectively. Cyclic compression modulated global Ca2+ signalling by increasing the percentage of cells exhibiting Ca2+ transients (population modulation) and/or reducing the rise and fall times of these transients (transient shape modulation). The frequency and strain rate of compression differentially modulated these Ca2+ signalling characteristics providing a potential mechanism through which chondrocytes may distinguish between different loading conditions. Treatment with apyrase, gadolinium and the P2 receptor blockers, suramin and basilen blue, significantly reduced the percentage of cells exhibiting Ca2+ transients following cyclic compression, such that the mechanically induced upregulation of Ca2+ signalling was completely abolished. Thus cyclic compression appears to activate a purinergic pathway involving the release of ATP followed by the activation of P2 receptors causing a combination of extracellular Ca2+ influx and intracellular Ca2+ release. Knowledge of this fundamental cartilage mechanotransduction pathway may lead to improved therapeutic strategies for the treatment of cartilage damage and disease.
This study investigates the use of gallium (Ga) based glass polyalkenoate cements (GPCs) as a possible alternative adhesive in sternal fixation, post sternotomy surgery. The glass series consists of a Control (CaO-ZnO-SiO2), and LGa-1 and LGa-2 which contain Ga at the expense of zinc (Zn) in 0.08 mol% increments. The additions of Ga resulted in increased working time (75 s to 137 s) and setting time (113 to 254 s). Fourier Transform Infrared (FTIR) analysis indicated that this was a direct result of increased unreacted poly(acrylic acid) (PAA) and the reduction of crosslink formation during cement maturation. LGa samples (0.16 wt % Ga) resulted in an altered ion release profile, particularly for 30 days analysis, with maximum Ca2+, Zn2+, Si4+ and Ga3+ ions released into the distilled water. The additions of Ga resulted in increased roughness and decreased contact angles during cement maturation. The presence of Ga has a positive effect on the compressive strength of the samples with strengths increasing over 10 MPa at 7 days analysis compared to the 1 day results. The additions of Ga had relatively no effect on the flexural strength. Tensile testing of bovine sterna proved that the LGa samples (0.16 wt % Ga) are comparable to the Control samples.
Bioactive glasses may function as antimicrobial delivery systems through the incorporation and subsequent release of therapeutic ions. The aim of this study was to evaluate the antimicrobial properties of a series of composite scaffolds composed of poly(octanediol citrate) with increased loads of a bioactive glass that releases zinc (Zn(2+)) and gallium (Ga(3+)) ions in a controlled manner. The antibacterial activity of these scaffolds was investigated against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. The ability of the scaffolds to release ions and the subsequent ingress of these ions into hard tissue was evaluated using a bovine bone model. Scaffolds containing bioactive glass exhibited antibacterial activity and this increased in vitro with higher bioactive glass loads; viable cells decreased to about 20 % for the composite scaffold containing 30 % bioactive glass. The Ga(3+) release rate increased as a function of time and Zn(2+) was shown to incorporate into the surrounding bone.
Degeneration of articular cartilage is often triggered by a small tissue crack. As cartilage structure and composition change with age, the mechanics of cracked cartilage may depend on the tissue age, but this relationship is poorly understood. Here, we investigated cartilage mechanics and crack deformation in immature and mature cartilage exposed to a full-thickness tissue crack using indentation testing and histology, respectively. When a cut was introduced, tissue cracks opened wider in the mature cartilage compared to the immature cartilage. However, the opposite occurred upon mechanical indentation over the cracked region. Functionally, the immature-cracked cartilages stress-relaxed faster, experienced increased tissue strain, and had reduced instantaneous stiffness, compared to the mature-cracked cartilages. Taken together, mature cartilage appears to withstand surface cracks and maintains its mechanical properties better than immature cartilage and these superior properties can be explained by the structure of their collagen fibrous network.
This study utilizes the technique of self-assembly to fabricate arrays of nanoislands on (001)-oriented yttria-stabilized zirconia single crystal substrates with miscut of 10° toward <110> direction. These self-assembled nanostructures were annealed at 1100°C for 5h upon doping with 10mol% gadolinium-doped ceria (GDC) by powder-suspension based method. X-Ray diffraction result showed that the miscut substrate after doping GDC was in the cubic phase. Energy dispersive X-ray (EDX) illustrates that the nanopatterned material contains all the elements from the GDC source and yttria-stabilized zirconia (YSZ) substrate. It also demonstrates a higher surface roughness and a more hydrophilic surface. The nanostructured materials were subsequently used for an in vitro study using a human fetal osteoblastic cell line (hFOB). An improved spreading, enhanced cell proliferation and up-regulated alkaline phosphatase activity (ALP) were observed on the nanopatterned substrates compared to the control substrates. Calcium deposits, which were stained positively by Alizarin Red S, appeared to be more abundant on the nanopatterned surfaces on day 7. The overall findings suggest that post fabrication treatment with surface modification such as creating a nanostructure (e.g. nanopatterns) can improve biocompatibility.
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.
Purpose:To investigate the feasibilty of using processed human amniotic membrane (HAM) to support the attachment and proliferation of chondrocytes in vitro which it turn can be utilised as a cell delivery vehicle in tissue engineering applications. Methods: Fresh HAM obtained from patients undergoing routine elective ceasarean sections was harvested., processed and dried using either freez drying (FD) or air drying (AD) methods prior to sterilisation by gamma irradiation. Isolated, processed and characterised rabbit autologous chondrolytes were seeded on processsed HAM and cultured for up to three weeks. Cell attachment and proliferation were examined qualitatively using inverted brightfield microcospy. Results: Processed HAM appeared to allow cell attachment when implanted with chrondocytes. Although cells seeded on AD and FD HAM did not appear to attach as strongly as those seeded on glycerol preserved intact human amniotic membrane, these cells to be proliferated in cell culture conditions. Conclusion: Prelimanary results show that processed HAM chondrocyte attachment and proliferation.
In this work, untreated bovine cortical bones (BCBs) were exposed to a range of heat treatments in order to determine at which temperature the apatite develops an optimum morphology comprising porous nano hydroxyapatite (nanoHAp) crystals. Rectangular specimens (10 mm × 10 mm × 3-5 mm) of BCB were prepared, being excised in normal to longitudinal and transverse directions. Specimens were sintered at up to 900 °C under ambient pressure in order to produce apatites by two steps sintering. The samples were characterized by thermogravimetric analysis, X-ray diffraction (XRD), and scanning electron microscopy (SEM) attached to an energy-dispersive X-ray spectroscopy detector. For the first time, morphology of the HAp particles was predicted by XRD, and it was verified by SEM. The results show that an equiaxed polycrystalline HAp particle with uniform porosity was produced at 900 °C. It indicates that a porous nanoHAp achieved by sintering at 900 °C can be an ideal candidate as an in situ scaffold for load-bearing tissue applications.
Biodegradable elastomers have clinical applicability due to their biocompatibility, tunable degradation and elasticity. The addition of bioactive glasses to these elastomers can impart mechanical properties sufficient for hard tissue replacement. Hence, a composite with a biodegradable polymer matrix and a bioglass filler can offer a method of augmenting existing tissue. This article reviews the applications of such composites for skeletal augmentation.
Based on the concept of LEGO toys, a fiber probe analytical platform (FPAP) was developed as a powerful diagnostic tool offering higher sensitivity in detection of infectious agents compared to established methods. Using the form and the function of LEGO toys, this protocol describes a fiber-based, 96-well plate, which suspends a new class of chemically-designed, electrospun fibers within the assay. This clamping strategy allows both sides of the developed fiber mats to interact with biomolecules within the assay thus benefiting from the tailored chemical and physical properties of these fiber-based bioreceptors in attracting the biomolecules to the surface. The fabrication method of FPAP involves one-step electrospinning of the chemically designed fibers, 3D printing of the LEGO-like probing segments, and assembly of the device followed by ELISA procedure. FPAP follows the same principles of operation as that of a conventional enzyme linked immunosorbent assay (ELISA), therefore, it can be run by lab technicians, expert in ELISA. FPAP was used for early diagnosis of Dengue fever and provided an 8-fold higher sensitivity while the limit of detection (LOD) was recorded to be in femto-gram per milliliter range which is significantly low when compared to other existing techniques or conventional assay. This platform allows different types of paper/fiber bio-receptive platforms to be incorporated within the design that promises simultaneous recognition of multiple infectious agents.
A mechanical-conditioning bioreactor has been developed to provide bi-axial loading to three-dimensional (3D) tissue constructs within a highly controlled environment. The computer-controlled bioreactor is capable of applying axial compressive and shear deformations, individually or simultaneously at various regimes of strain and frequency. The reliability and reproducibility of the system were verified through validation of the spatial and temporal accuracy of platen movement, which was maintained over the operating length of the system. In the presence of actual specimens, the system was verified to be able to deliver precise bi-axial load to the specimens, in which the deformation of every specimen was observed to be relatively homogeneous. The primary use of the bioreactor is in the culture of chondrocytes seeded within an agarose hydrogel while subjected to physiological compressive and shear deformation. The system has been designed specifically to permit the repeatable quantification and characterisation of the biosynthetic activity of cells in response to a wide range of short and long term multi-dimensional loading regimes.
The investigation of patellar tendon reflex involves development of a reflex hammer holder, kinematic data collection and analysis of patellar reflex responses using motion analysis techniques. The main aim of this research is to explore alternative means of assessing reflexes as a part of routine clinical diagnosis. The motion analysis system was applied to provide quantitative data which is a more objective measure of the patellar tendon reflex. Kinematic data was collected from 28 males and 22 females whilst subjected to a knee jerk test. Further analysis of kinematic data was performed to predict relationships which might affect the patellar tendon reflex. All subjects were seated on a high stool with their legs hanging freely within the capture volume of the motion analysis system. Knee jerk tests were applied to all subjects, on both sides of the leg, by eliciting hypo, hyper, and normal reflexes. An additional reinforcement technique called the Jendrassik manoeuvre was also performed under the same conditions to elicit a normal patellar tendon reflex. The comparison of reflex response between genders showed that female subjects generally had a greater response compared to males. However, the difference in reflex response between the left leg and the right leg was not significant. Tapping strength to elicit a hyper-reflex produced greater knee-jerk compared to the normal clinical tapping strength. All results were in agreement with clinical findings and results found by some early researchers.
C-type natriuretic peptide (CNP) has been demonstrated in human and mouse models to play critical roles in cartilage homeostasis and endochondral bone formation. Indeed, targeted inactivation of the genes encoding CNP results in severe dwarfism and skeletal defects with a reduction in growth plate chondrocytes. Conversely, cartilage-specific overexpression of CNP was observed to rescue the phenotype of CNP deficient mice and significantly enhanced bone growth caused by growth plate expansion. In vitro studies reported that exogenous CNP influenced chondrocyte differentiation, proliferation and matrix synthesis with the response dependent on CNP concentration. The chondroprotective effects were shown to be mediated by natriuretic peptide receptor (Npr)2 and enhanced synthesis of cyclic guanosine-3',5'-monophosphate (cGMP) production. Recent studies also showed certain homeostatic effects of CNP are mediated by the clearance inactivation receptor, Npr3, highlighting several mechanisms in maintaining tissue homeostasis. However, the CNP signalling systems are complex and influenced by multiple factors that will lead to altered signalling and tissue dysfunction. This review will discuss the differential role of CNP signalling in regulating cartilage and bone homeostasis and how the pathways are influenced by age, inflammation or sex. Evidence indicates that enhanced CNP signalling may prevent growth retardation and protect cartilage in patients with inflammatory joint disease.
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.
The role for mechanical stimulation in the control of cell fate has been previously proposed, suggesting that there may be a role of mechanical conditioning in directing mesenchymal stromal cells (MSCs) towards specific lineage for tissue engineering applications. Although previous studies have reported that calcium signalling is involved in regulating many cellular processes in many cell types, its role in managing cellular responses to tensile loading (mechanotransduction) of MSCs has not been fully elucidated. In order to establish this, we disrupted calcium signalling by blocking stretch-activated calcium channel (SACC) in human MSCs (hMSCs) in vitro. Passaged-2 hMSCs were exposed to cyclic tensile loading (1 Hz + 8% for 6, 24, 48, and 72 hours) in the presence of the SACC blocker, gadolinium. Analyses include image observations of immunochemistry and immunofluorescence staining from extracellular matrix (ECM) production, and measuring related tenogenic and apoptosis gene marker expression. Uniaxial tensile loading increased the expression of tenogenic markers and ECM production. However, exposure to strain in the presence of 20 μM gadolinium reduced the induction of almost all tenogenic markers and ECM staining, suggesting that SACC acts as a mechanosensor in strain-induced hMSC tenogenic differentiation process. Although cell death was observed in prolonged stretching, it did not appear to be apoptosis mediated. In conclusion, the knowledge gained in this study by elucidating the role of calcium in MSC mechanotransduction processes, and that in prolonged stretching results in non-apoptosis mediated cell death may be potential useful for regenerative medicine applications.
(1) Background: Stem cells in combination with scaffolds and bioactive molecules have made significant contributions to the regeneration of damaged bone tissues. A co-culture system can be effective in enhancing the proliferation rate and osteogenic differentiation of the stem cells. Hence, the aim of this study was to investigate the osteogenic differentiation of human adipose derived stem cells when co-cultured with human osteoblasts and seeded on polycaprolactone (PCL):hydroxyapatite (HA) scaffold; (2) Methods: Human adipose-derived stem cells (ASC) and human osteoblasts (HOB) were seeded in three different ratios of 1:2, 1:2 and 2:1 in the PCL-HA scaffolds. The osteogenic differentiation ability was evaluated based on cell morphology, proliferation rate, alkaline phosphatase (ALP) activity, calcium deposition and osteogenic genes expression levels using quantitative RT-PCR; (3) Results: The co-cultured of ASC/HOB in ratio 2:1 seeded on the PCL-HA scaffolds showed the most positive osteogenic differentiation as compared to other groups, which resulted in higher ALP activity, calcium deposition and osteogenic genes expression, particularly Runx, ALP and BSP. These genes indicate that the co-cultured ASC/HOB seeded on PCL-HA was at the early stage of osteogenic development; (4) Conclusions: The combination of co-culture system (ASC/HOB) and PCL-HA scaffolds promote osteogenic differentiation and early bone formation.