Differences in morphogenesis of 3D cultured primary human osteoblasts under static and microfluidic growth conditions

As information on osteoblast mechanosensitivity response to biomechanical cues in three-dimensional (3D) in vitro microenvironments is sparse, the present study compared morphogenesis of primary human alveolar bone osteoblasts (PHABO) under microchip-based 3D-static conditions, and 3D-fluid flow-mediated biomechanical stimulation in perfusion bioreactors. Discrimination of the respective microenvironment by differential morphogenesis was evident from fluid flow-induced PHABO reorganization into rotund bony microtissue, comprising more densely packed multicellular 3D-aggregates, while viability of microtissues was flow rate dependent. Time-lapse microscopy and simple modeling of biomechanical conditions revealed that physiologically relevant fluid flow-mediated PHABO stimulation was associated with formation of mulberry-like PHABO aggregates within the first 24 h. Differential extracellular matrix deposition patterns and gene expression modulation in PHABO aggregates at day 7 further indicates progressive osteoblast differentiation exclusively in perfusion culture-developed bony microtissues. The results of our study strongly suggest PHABO morphogenesis as discriminator of microenvironmental growth conditions, which in case of the microfluidic 3D microchip-bioreactor are substantiated by triggering in vitro bone microtissue formation concomitant with progressive osteoblastic differentiation. Such microtissue outcomes provide unique insight for mechanobiological studies in response to biomechanical fluid flow cues, and clinically appear promising for in vitro PHABO preconditioning, enabling innovative bone augmentation procedures.     

Control of Mammalian Cell and Bacteria Adhesion on Substrates Micropatterned with Poly(ethylene glycol) Hydrogels

A simple method for controlling the spatial positioning of mammalian cells and bacteria on substrates using patterned poly(ethylene glycol) (PEG) hydrogel microstructures is described. These microstructures were fabricated using photolithography on silicon, glass or poly (dimethylsiloxane) (PDMS) surfaces modified with a 3-(trichlorosilyl) propyl methacrylate (TPM) monolayer. During the photogelation reaction, the resulting hydrogel microstructures were covalently bound to the substrate via the TPM monolayer and did not detached from the substrate upon hydration. For mammalian cell patterning, microwell arrays of different dimensions were fabricated. These microwells were composed of hydrophilic PEG hydrogel walls surrounding hydrophobic TPM floors inside the microwells. Murine 3T3 fibroblasts and transformed hepatocytes were shown to selectively adhere to the TPM monolayer inside the microwells, maintaining their viability, while adherent cells were not present on the hydrogel walls. The number of cells inside one microwell could be controled by changing the lateral dimension of the microwells, thus allowing only a single cell per microwell if desired. In the case of 30×30 m microwells, as many as 400 microwells were fabricated in 1 mm². In addition, PEG hydrogel microstructures were also shown to effectively resist the adhesion of bacteria such as Escherichia coli.     

Human umbilical cord-blood-derived mesenchymal stem cell can improve the clinical outcome and Joint space width after high tibial osteotomy

BackgroundThere is limited study about the human umbilical cord-blood derived mesenchymal stem cell (hUCB-MSC) cartilage regeneration procedures combined with high tibial osteotomy (HTO). We compared the clinical and radiological results of hUCB-MSC cartilage regeneration procedures combined with HTO to those of microfracture with HTO.MethodsFrom August 2017 to December 2018, HTO patients with International Cartilage Regeneration and Joint Preservation Society (ICRS) grade IV cartilage defects over 200 mm² on medial femoral condyle (MFC) were enrolled. For comparison, all participants were divided into two groups: those who had undergone an hUCB-MSC induced cartilage regeneration procedure (group MSC) and those with microfractures only (group C, controls). Clinically, Hospital for Special Surgery (HSS), International Knee Documentation Committee (IKDC), and Lysholm scores were evaluated post-operatively at 18 months. Radiologically, mechanical axis (MA) and joint space width (JSW) were evaluated.ResultsA total of 100 knees were enrolled (43 in group MSC, 57 in group C). The IKDC score in group MSC (69) was better than that in group C (62; P < 0.05). The JSW increment in the MSC group (0.6 mm) was more than that in group C (0.1 mm; P < 0.05). No patient developed nonunion, correction loss, or arthroplasty conversion.ConclusionhUCB-MSCs can improve clinical outcome and JSW better than microfracture only in HTO patients.     

A microwell pattern for C17.2 cell aggregate formation with concave cylindrical surface induced cell peeling

We have developed a polydimethylsiloxane (PDMS) pattern with arrays of microwells for the formation of multicellular aggregates by C17.2 neural stem cells. Upon interfacing with the patterns, the neural stem cells would firstly attach to the microwell sidewalls, forming cellular strips on day 1 after plating. For channel connected microwells, cellular strips on the concave semi-cylindrical sidewall surfaces continued among wells and through channels, followed by strip peeling due to prestress arising from actin filaments and assembly of suspending cellular aggregates within the microwells in the following 1–2 days. Our results also suggested that a small microwell diameter of 80 and 100 μm and a narrow channel width of 20 μm would facilitate the aggregate formation among the structural dimensions tested. Finite element method (FEM) simulation revealed that cellular strips on the semi-cylindrical sidewall surfaces peeled under significantly smaller prestresses (critical peeling prestress, CPP), than cells on flat substrates. However, the CPP by itself failed to fully account for the difference in aggregate inducing capability among the patterns addressed, suggesting cell growth behaviors might play a role. This study thus justified the current patterning method as a unique and practical approach for establishing 3D neural stem cell-based assay platforms.     

Mesenchymal stem cell-collagen microspheres for articular cartilage repair: Cell density and differentiation status

Mesenchymal stem cells (MSC) hold promise for cartilage repair. A microencapsulation technique was previously established to entrap MSC in collagen microspheres, and the collagen fibrous meshwork was found to be an excellent scaffold for supporting MSC survival, growth and differentiation. This study investigates the importance of cell density and differentiation status of MSC–collagen microspheres in cartilage repair. MSC were isolated from rabbit bone marrow and encapsulated in collagen microspheres. The effects of pre-differentiating the encapsulated MSC into chondrogenic lineages and different cell densities on cartilage repair were investigated in rabbits. Implantation of undifferentiated collagen–MSC microspheres formed hyaline-like cartilage rich in type II collagen and glycosaminoglycans (GAG) at 1 month post-implantation. By 6 months, hyaline cartilage rich in type II collagen and GAG, but negative for type I collagen, and partial zonal organization were found in both undifferentiated and chondrogenically differentiated groups in the high cell density group. The undifferentiated group and high cell density group significantly improved the O’Driscoll histological score. Moreover, the undifferentiated group significantly increased the GAG content. The mechanically differentiated group showed stiffer but thinner cartilage, while the undifferentiated group showed thicker but softer cartilage compared with their respective contra-lateral controls. This work suggests that a higher local cell density favors cartilage regeneration, regardless of the differentiation status of MSC, while the differentiation status of MSC does significantly affect regeneration outcomes.     

Intra-articular injection of human synovium-derived mesenchymal stem cells in beagles with surgery-induced osteoarthritis

BackgroundRecently, cell-based tissue engineering approaches using mesenchymal stem cells (MSCs) have been used to treat osteoarthritis (OA). However, the efficacy of human synovium-derived MSCs (hSD-MSCs) has not yet been tested in a canine model of OA. The purpose of this study was to investigate the therapeutic effects of intra-articular hSD-MSC injections in a canine OA model.MethodsSixty beagles underwent surgical manipulation to induce OA and received intra-articular injection 4 weeks after surgery. The dogs were divided into five groups (n = 12) according to the injection material: G1, sham group; G2, control group injected with phosphate-buffered saline; G3, G4, and G5, experimental groups injected with different hSD-MSC dosages (G3, 2.4 × 10⁶ cells; G4, 4.8 × 10⁶ cells; G5, 9.6 × 10⁶ cells). Magnetic resonance imaging (MRI) and histopathological and immunohistochemical examinations were performed 6 and 24 weeks after injection.ResultsMRI revealed significant improvements in synovitis 24 weeks after injection in the hSD-MSC-injected groups (G3–G5). Histopathologic analyses showed that cartilage structure and proteoglycan staining were also significantly improved in these groups (G3–G5) 6 weeks after injection and improved further after 24 weeks. Immunohistochemical analysis revealed significant differences in the levels of collagen types I and II between the hSD-injected groups (G3–G5), indicating a similar extracellular matrix (ECM) composition to naïve articular cartilage.ConclusionOur study demonstrated for the first time that intra-articular hSD-MSC injection ameliorates the progression of canine OA by restoring cartilage, promoting ECM synthesis, and inhibiting the inflammatory response.     

Ice-lithographic fabrication of concave microwells and a microfluidic network

We describe a novel method to produce concave microwells utilizing solid–liquid phase change. This method, named ‘ice-lithography’, does not require any lithographic processes and consists of a few simple steps that yield multiple concave microwells. We demonstrated that the shape and size of the microwells can be controlled by varying substrates and vapor-collection time. Patterned wells with sizes in the range of 10μm to several millimeters in diameter could be produced. Additionally, we fabricated a uniformly aligned concave microwell pattern and a microfluidic network. Ice-lithography has potential biological and biomedical applications in areas such as the fabrication of cell docking devices and microbioreactors as well as the formation of uniformly sized embryoid bodies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Micro/nanoscale technologies for the development of hormone-expressing islet-like cell clusters

Insulin-expressing islet-like cell clusters derived from precursor cells have significant potential in the treatment of type-I diabetes. Given that cluster size and uniformity are known to influence islet cell behavior, the ability to effectively control these parameters could find applications in the development of anti-diabetic therapies. In this work, we combined micro and nanofabrication techniques to build a biodegradable platform capable of supporting the formation of islet-like structures from pancreatic precursors. Soft lithography and electrospinning were used to create arrays of microwells (150-500 μm diameter) structurally interfaced with a porous sheet of micro/nanoscale polyblend fibers (~0.5-10 μm in cross-sectional size), upon which human pancreatic ductal epithelial cells anchored and assembled into insulin-expressing 3D clusters. The microwells effectively regulated the spatial distribution of the cells on the platform, as well as cluster size, shape and homogeneity. Average cluster cross-sectional area (~14000-17500 μm(2)) varied in proportion to the microwell dimensions, and mean circularity values remained above 0.7 for all microwell sizes. In comparison, clustering on control surfaces (fibers without microwells or tissue culture plastic) resulted in irregularly shaped/sized cell aggregates. Immunoreactivity for insulin, C-peptide and glucagon was detected on both the platform and control surfaces; however, intracellular levels of C-peptide/cell were ~60 % higher on the platform.     

Chondrogenesis and Integration of Mesenchymal Stem Cells Within an <Emphasis Type="Italic">In</Emphasis> <Emphasis Type="Italic">Vitro</Emphasis> Cartilage Defect Repair Model

Integration of repair tissue is a key indicator of the long-term success of cell-based therapies for cartilage repair. The objective of this study was to compare the in vitro chondrogenic differentiation and integration of agarose hydrogels seeded with either chondrocytes or bone marrow-derived mesenchymal stem cells (MSCs) in defects created in cartilage explants. Chondrocytes and MSCs were isolated from porcine donors, suspended in 2% agarose and then injected into cylindrical defects within the explants. These constructs were maintained in a chemically defined medium supplemented with 10 ng/mL of TGF-β3. Cartilage integration was assessed by histology and mechanical push-out tests. After 6 weeks in culture, chondrocyte-seeded constructs demonstrated a higher integration strength (64.4 ± 8.3 kPa) compared to MSC-seeded constructs (22.7 ± 5.9 kPa). Glycosaminoglycan (GAG) (1.27 ± 0.3 vs. 0.19 ± 0.03 kPa) and collagen (0.31 ± 0.08 vs. 0.09 ± 0.01 kPa) accumulation in chondrocyte-seeded constructs was greater than that measured in the MSC-seeded group. The GAG, collagen, and DNA content of both chondrocyte- and MSC-seeded hydrogels cultured in cartilage explants was significantly lower than control constructs cultured in free swelling conditions. The results of this study suggest that the explant model may constitute a more rigorous in vitro test to assess MSC therapies for cartilage defect repair.     

Treatment of Graft versus Host Disease with Mesenchymal Stromal Cells: A Phase I Study on 40 Adult and Pediatric Patients

This phase I multicenter study was aimed at assessing the feasibility and safety of intravenous administration of third party bone marrow–derived mesenchymal stromal cells (MSC) expanded in platelet lysate in 40 patients (15 children and 25 adults), experiencing steroid-resistant grade II to IV graft-versus-host disease (GVHD). Patients received a median of 3 MSC infusions after having failed conventional immunosuppressive therapy. A median cell dose of 1.5 × 10⁶/kg per infusion was administered. No acute toxicity was reported. Overall, 86 adverse events and serious adverse events were reported in the study, most of which (72.1%) were of infectious nature. Overall response rate, measured at 28 days after the last MSC injection, was 67.5%, with 27.5% complete response. The latter was significantly more frequent in patients exhibiting grade II GVHD as compared with higher grades (61.5% versus 11.1%, P = .002) and was borderline significant in children as compared with adults (46.7 versus 16.0%, P = .065). Overall survival at 1 and 2 years from the first MSC administration was 50.0% and 38.6%, with a median survival time of 1.1 years. In conclusion, MSC can be safely administered on top of conventional immunosuppression for steroid resistant GVHD treatment. Eudract Number 2008-007869-23, NCT01764100.     

Vestibular thresholds for yaw rotation about an earth-vertical axis as a function of frequency

Perceptual direction detection thresholds for yaw rotation about an earth-vertical axis were measured at seven frequencies (0.05, 0.1, 0.2, 0.5, 1, 2, and 5 Hz) in seven subjects in the dark. Motion stimuli consisted of single cycles of sinusoidal acceleration and were generated by a motion platform. An adaptive two-alternative categorical forced-choice procedure was used. The subjects had to indicate by button presses whether they perceived yaw rotation to the left or to the right. Thresholds were measured using a 3-down, 1-up staircase paradigm. Mean yaw rotation velocity thresholds were 2.8 deg s⁻¹ for 0.05 Hz, 2.5 deg s⁻¹ for 0.1 Hz, 1.7 deg s⁻¹ for 0.2 Hz, 0.7 deg s⁻¹ for 0.5 Hz, 0.6 deg s⁻¹ for 1 Hz, 0.4 deg s⁻¹ for 2 Hz, and 0.6 deg s⁻¹ for 5 Hz. The results show that motion thresholds increase at 0.2 Hz and below and plateau at 0.5 Hz and above. Increasing velocity thresholds at lower frequencies qualitatively mimic the high-pass characteristics of the semicircular canals, since the increase at 0.2 Hz and below would be consistent with decreased gain/sensitivity observed in the VOR at lower frequencies. In fact, the measured dynamics are consistent with a high pass filter having a threshold plateau of 0.71 deg s^-1 and a cut-off frequency of 0.23 Hz, which corresponds to a time constant of approximately 0.70 s. These findings provide no evidence for an influence of velocity storage on perceptual yaw rotation thresholds.

Efficient Dielectrophoretic Patterning of Embryonic Stem Cells in Energy Landscapes Defined by Hydrogel Geometries

In this study, we have developed an integrated microfluidic platform for actively patterning mammalian cells, where poly(ethylene glycol) (PEG) hydrogels play two important roles as a non-fouling layer and a dielectric structure. The developed system has an embedded array of PEG microwells fabricated on a planar indium tin oxide (ITO) electrode. Due to its dielectric properties, the PEG microwells define electrical energy landscapes, effectively forming positive dielectrophoresis (DEP) traps in a low-conductivity environment. Distribution of DEP forces on a model cell was first estimated by computationally solving quasi-electrostatic Maxwell’s equations, followed by an experimental demonstration of cell and particle patterning without an external flow. Furthermore, efficient patterning of mouse embryonic stem (mES) cells was successfully achieved in combination with an external flow. With a seeding density of 10⁷ cells/mL and a flow rate of 3 μL/min, trapping of cells in the microwells was completed in tens of seconds after initiation of the DEP operation. Captured cells subsequently formed viable and homogeneous monolayer patterns. This simple approach could provide an efficient strategy for fabricating various cell microarrays for applications such as cell-based biosensors, drug discovery, and cell microenvironment studies.     

Matrix-driven formation of mesenchymal stem cell–extracellular matrix microtissues on soft alginate hydrogels

Mesenchymal stem cells (MSCs) can be made to rearrange into microtissues in response to specific matrix cues, a process that depends on a balance between cell–matrix and cell–cell interactions. The effect of such cues, and especially their interplay, is still not fully understood, particularly in three-dimensional (3-D) systems. Here, the behaviour of human MSCs cultured within hydrogel matrices with tailored stiffness and composition was evaluated. MSC aggregation occurred only in more compliant matrices (G′ ⩽ 120 Pa), when compared to stiffer ones, both in the presence and in the absence of matrix-bound arginine–glycine–aspartic acid cell–adhesion ligands (RGD; 0, 100 and 200 μM). Fibronectin assembly stabilized cell–cell contacts within aggregates, even in non-adhesive matrices. However, MSCs were able to substantially contract the artificial matrix only when RGD was present. Moreover, compliant matrices facilitated cell proliferation and provided an environment conducive for MSC osteogenic differentiation, even without RGD. Cell interactions with the original matrix became less important as time progressed, while the de novo-produced extracellular matrix became a more critical determinant of cell fate. These data provide further insights into the mechanisms by which MSCs sense their microenvironment to organize into tissues, and provide new clues to the design of cell-instructive 3-D matrices.     

Sequential Third-Party Mesenchymal Stromal Cell Therapy for Refractory Acute Graft-versus-Host Disease

We evaluated the feasibility, safety, and efficacy of the administration of 4 sequential doses (intravenously administered on days 1, 4, 11, and 18) of cryopreserved bone marrow–derived mesenchymal stromal cells (MSC) expanded with platelet lysate and obtained from third-party donors as a second-line treatment for steroid-refractory acute graft-versus-host (aGVHD) disease in a series of 25 patients. All patients received at least 2 doses of MSC, whereas 21 received 3 doses and 18 received the initially planned 4 doses. Because of the achievement of partial response, 4 patients received additional doses of MSC. Median single cell dose administered was 1.1 × 10⁶ MSC/kg of recipient body weight. There were no adverse events related to the MSC infusion in the 99 procedures performed, with the exception of a cardiac ischemic event that occurred twice in a patient with prior history of cardiac ischemia. Response to MSC at 60 days after the first dose was evaluable in 24 patients. Seventeen patients (71%) responded (11 complete and 6 partial responses), with a median time to response of 28 days after the first MSC dose, whereas 7 patients did not respond. In summary, we can conclude that sequential cryopreserved third-party MSC therapy administered on days 1, 4, 11, and 18 is a safe procedure for patients with steroid-refractory aGVHD. This strategy may provide a high rate of overall responses of aGVHD with a low toxicity profile.     

Detection and evaluation of initial cartilage pathology in man: A comparison between MRT,arthroscopy and near-infrared spectroscopy (NIR) in their relation to initial knee pain

Background and aims: MRI and arthroscopy are important methods in the evaluation of cartilage pathology. But frequently initial changes of cartilage in combination with chronic knee pain cannot be detected by employing these two methods. Better diagnostic tools for the detection of the early stages of osteoarthritis (OA) are required. The objective of this study was to show that near-infrared spectroscopy (NIRS) can be incorporated into routine arthroscopy to improve detection and assessment of the initial cartilage pathology. Furthermore correlations between findings in MRI, arthroscopy and NIRS in patients with initial symptoms of OA have studied. Methods: Patients (n = 21, 12 women, 9 men, age: 15–59 years, mean 34.19 years) with knee pain lasting for at least half a year without any trauma of the knee in their history were interviewed (body weight, smoking behaviour) and clinically evaluated using the Knee Injury and Osteoarthritis Outcome Score (KOOS). Also serum parameters (cholesterol, lipids) were analysed, conventional X-rays in three directions (evaluated according to Kellgren and Lawrence) and MRI (evaluation of cartilage damage according to the ICRS-score) were performed preoperatively in all patients. During subsequent arthroscopy cartilage damage was evaluated according to the ICRS-score. In addition the spectral reflection of cartilage was investigated in all knees using a special micro-glass-fiber probe in the near-infrared light region (spectral range between 1150 and 1475 nm). To characterize relations between the investigated parameters the Spearman's rank correlation coefficient was used. Inter-observer variance was calculated employing the Cohens–Kappa-test. Results: MRI demonstrated a strong inter-observer variance with no significant correlations to other parameters. The same was observed for arthroscopic findings. Only NIRS showed significant correlations with three out of five KOOS subscores. Within the general parameters only smoking behaviour showed a significant correlation with two of the KOOS-scores. NIRS therefore seemed to be a sensitive diagnostic tool in detection of initial pathology in human cartilage. The additional necessary time for the spectroscopic investigation as part of the routine arthroscopy ranged between 3 and 7 min (mean: 4 min 18 s). Conclusion: Particularly for early-stage cartilage lesions (ICRS 0/I) MRI and arthroscopy have rather low predictive value. The inter-observer variance is very high (Cohens–Kappa < 0.4). Correlations found between NIRS and KOOS suggest that NIRS potentially can be used for detection of initial cartilage pathology and may be helpful in the evaluation of the benefit of different medical or surgical interventions at early-stage of articular cartilage damage.     

Semi-interpenetrating networks of hyaluronic acid in degradable PEG hydrogels for cartilage tissue engineering

Hydrolytically biodegradable poly(ethylene glycol) (PEG) hydrogels offer a promising platform for chondrocyte encapsulation and tuning degradation for cartilage tissue engineering, but offer no bioactive cues to encapsulated cells. This study tests the hypothesis that a semi-interpenetrating network of entrapped hyaluronic acid (HA), a bioactive molecule that binds cell surface receptors on chondrocytes, and crosslinked degradable PEG improves matrix synthesis by encapsulated chondrocytes. Degradation was achieved by incorporating oligo (lactic acid) segments into the crosslinks. The effects of HA molecular weight (MW) (2.9 × 10⁴ and 2 × 10⁶ Da) and concentration (0.5 and 5 mg g⁻¹) were investigated. Bovine chondrocytes were encapsulated in semi-interpenetrating networks and cultured for 4 weeks. A steady release of HA was observed over the course of the study with 90% released by 4 weeks. Incorporation of HA led to significantly higher cell numbers throughout the culture period. After 8 days, HA increased collagen content per cell, increased aggrecan-positive cells, while decreasing the deposition of hypertrophic collagen X, but these effects were not sustained long term. Measuring total sulfated glycosaminoglycan (sGAG) and collagen content within the constructs and released to the culture medium after 4 weeks revealed that total matrix synthesis was elevated by high concentrations of HA, indicating that HA stimulated matrix production although this matrix was not retained within the hydrogels. Matrix-degrading enzymes were elevated in the low-, but not the high-MW HA. Overall, incorporating high-MW HA into degrading hydrogels increased chondrocyte number and sGAG and collagen production, warranting further investigations to improve retention of newly synthesized matrix molecules.     

Transfection and intracellular trafficking characteristics for poly(amidoamine)s with pendant primary amine in the delivery of plasmid DNA to bone marrow stromal cells

Poly(amidoamine)s with pendant primary amine (polymer 1a–1c) were evaluated as in vitro non-viral gene delivery vectors for bone marrow stromal cells (BMSCs). The cytotoxicity of these poly(amidoamine)s, measured by MTT assay, increased with increasing length of side chain, however, they were less toxic than branched polyethylenimine (PEI) 25 kDa. Using pGL-3 and pEGFP-C1 as luciferase gene and green fluorescent protein (GFP) gene, among all polycations including polymer 1a–1c and PEI, polymer 1b at optimal N/P ratio showed highest luciferase expression (1.92 × 10⁸ RLU/mg protein) as well as percentage of cells expressing GFP (29.01 ± 2.33%). For all polycations, intracellular trafficking of Cy3-labelled plasmid DNA (pDNA) was similar. Fluorescent particles attached to cell membrane at 0.5 h after adding the polycation/DNA complexes, aggregated in cytoplasm after 2 h, and then stayed around the perinuclear region after 4 h. pDNA nuclear localization appeared at 4 h post-transfection, but much more pDNA entered into nucleus at 24 h. At high N/P ratio, polymer 1a–1c could deliver pDNA into 70–80% of BMSCs after 24 h transfection, however, labelled pDNA was observed in only 4–25% of cells at the same time. Compared to PEI, polymer 1b showed comparable or even higher percentage of pDNA uptake and nuclear localization. We concluded that poly(amidoamine)s with pendant primary amine, especially polymer 1b, are new kind of promising candidates of less toxic and highly efficient non-viral gene delivery vectors for BMSCs.     

Nerve guides manufactured from photocurable polymers to aid peripheral nerve repair

The peripheral nervous system has a limited innate capacity for self-repair following injury, and surgical intervention is often required. For injuries greater than a few millimeters autografting is standard practice although it is associated with donor site morbidity and is limited in its availability. Because of this, nerve guidance conduits (NGCs) can be viewed as an advantageous alternative, but currently have limited efficacy for short and large injury gaps in comparison to autograft. Current commercially available NGC designs rely on existing regulatory approved materials and traditional production methods, limiting improvement of their design. The aim of this study was to establish a novel method for NGC manufacture using a custom built laser-based microstereolithography (μSL) setup that incorporated a 405 nm laser source to produce 3D constructs with ∼50 μm resolution from a photocurable poly(ethylene glycol) resin. These were evaluated by SEM, in vitro neuronal, Schwann and dorsal root ganglion culture and in vivo using a thy-1-YFP-H mouse common fibular nerve injury model. NGCs with dimensions of 1 mm internal diameter × 5 mm length with a wall thickness of 250 μm were fabricated and capable of supporting re-innervation across a 3 mm injury gap after 21 days, with results close to that of an autograft control. The study provides a technology platform for the rapid microfabrication of biocompatible materials, a novel method for in vivo evaluation, and a benchmark for future development in more advanced NGC designs, biodegradable and larger device sizes, and longer-term implantation studies.     

A novel carboxymethyl chitosan–quantum dot-based intracellular probe for Zn2+ ion sensing in prostate cancer cells

In this paper, we fabricated novel carboxymethyl chitosan-coated CdTe quantum dots (CMC–CdTe QDs) via the electrostatic interaction between amino groups in the carboxymethyl chitosan polymeric chains and carboxyl groups of the CdTe QDs. Carboxymethyl chitosan on the surface of CdTe QDs had strong binding ability with Zn²⁺, resulting in the obvious enhancement of the photoluminescence of CdTe QDs. The photoluminescence intensity of CMC–CdTe QDs probe was proportional to the concentration of Zn²⁺ in the range of 5.0 × 10⁻⁶ to 5.0 × 10⁻³ mol l⁻¹. The detection limit for Zn²⁺ was 4.5 × 10⁻⁶ mol l⁻¹. The experimental results indicate that the CMC–CdTe QDs possess favorable cell compatibility, good sensitivity and selectivity for intracellular Zn²⁺ sensing, and are promising candidates for cellular imaging and sensing in prostate cancer cells. The present study also provides an approach for the further development of nanoprobes dedicated to intracellular sensing.     

A computational and experimental study inside microfluidic systems: the role of shear stress and flow recirculation in cell docking

In this paper, microfluidic devices containing microwells that enabled cell docking were investigated. We theoretically assessed the effect of geometry on recirculation areas and wall shear stress patterns within microwells and studied the relationship between the computational predictions and experimental cell docking. We used microchannels with 150 μm diameter microwells that had either 20 or 80 μm thickness. Flow within 80 μm deep microwells was subject to extensive recirculation areas and low shear stresses (<0.5 mPa) near the well base; whilst these were only presented within a 10 μm peripheral ring in 20 μm thick microwells. We also experimentally demonstrated that cell docking was significantly higher (p < 0.01) in 80 μm thick microwells as compared to 20 μm thick microwells. Finally, a computational tool which correlated physical and geometrical parameters of microwells with their fluid dynamic environment was developed and was also experimentally confirmed.