generation-size-controlled-poly-ethylene-glycol-diacrylate-droplets

Generation of Size-controlled Poly (ethylene Glycol) Diacrylate Droplets <em>via </em>Semi-3-Dimensional Flow Focusing Microfluidic Devices

Uniform and size-controllable poly (ethylene glycol) diacrylate (PEGDA) droplets could be produced via the flow focusing process in a microfluidic device. ...

Tsinghua University

Corneal blindness caused by limbal stem cell deficiency (LSCD) is a prevailing disorder worldwide. Clinical outcomes for LSCD therapy using amniotic membrane (AM) are unpredictable. Hydrogels can eliminate limitations of standard therapy for LSCD, because they present all the advantages of AM (i.e. biocompatibility, inertness and a biodegradable structure) but unlike AM, they are structurally uniform and can be easily manipulated to alter mechanical and physical properties. Hydrogels can be delivered with minimum trauma to the ocular surface and do not require extensive serological screening before clinical application. The hydrogel structure is also amenable to modifications which direct stem cell fate. In this focussed review we highlight hydrogels as biomaterial substrates which may replace and/or complement AM in the treatment of LSCD.

Towards the use of hydrogels in the treatment of limbal stem cell deficiency.

Collagen has excellent biocompatibility, is biodegradable, and possesses low immunogenicity. Therefore, this protein is a very suitable substrate for the formation of a corneal scaffold for therapeutic use. The highly hydrated nature of conventional collagen gels, however, results in a gel that is structurally weak and difficult to manipulate. In this chapter, we describe a novel method to cultivate limbal epithelial cells (LEC) on a compressed collagen scaffold. The compressed collagen scaffold can be rapidly constructed using a cell-independent process, which produces dense and mechanically strong collagen constructs with controllable microscale features.We embedded corneal keratocytes in a collagen gel, which we subsequently compressed and coated with laminin. The resulting construct supported the physiological morphology and stratification of LEC. The expression of a specific marker for differentiated LEC, cytokeratin 3 (CK3), and a marker for undifferentiated LEC, cytokeratin 14 (CK14), were similar in LEC expanded on both the compressed collagen construct and the leading conventional scaffold, denuded amniotic membrane (AM). We therefore demonstrate that a laminin-coated, compressed collagen gel containing keratocytes can support LEC expansion, stratification, and differentiation to a degree that is comparable to denuded AM. Our novel compressed collagen/keratocyte construct has potential for use as a tissue-engineered artificial cornea.

The formation of a tissue-engineered cornea using plastically compressed collagen scaffolds and limbal stem cells.

Scaffolds are of great importance to the success of tissue engineering. Poly(l-lactide-co-glycolide) acid is one of the most commonly used biopolymers. This study develops a novel low-temperature deposition machine using screw extrusion to fabricate poly(l-lactide-co-glycolide) acid scaffolds. The screw extrusion process of poly(l-lactide-co-glycolide) acid is analysed, and the relationship between flow rate and processing parameters is examined. This relationship provides guidelines for optimizing the processing parameters. The major components and design strategy of the fabrication system are introduced. Measures are proposed to control the leakage of materials, and optimal processing parameters are determined. The machine is also equipped with a double-screw extrusion nozzle system; preliminary results demonstrate its capacity to fabricate gradient scaffolds. Porous structure characterization using mercury porosimetry demonstrates that the fabrication system is able to fabricate poly(l-lactide-co-glycolide) acid scaffolds that are both macroporous and microporous.

Development of a novel low-temperature deposition machine using screw extrusion to fabricate poly(l-lactide-co-glycolide) acid scaffolds.

Alginate hydrogel is a popular biologically inert material that is widely used in 3D bioprinting, especially in extrusion-based printing. However, the printed cells in this hydrogel could not degrade the surrounding alginate gel matrix, causing them to remain in a poorly proliferating and non-differentiating state. Here, we report a novel study of the 3D printing of human corneal epithelial cells (HCECs)/collagen/gelatin/alginate hydrogel incubated with a medium containing sodium citrate to obtain degradation-controllable cell-laden tissue constructs. The 3D-printed hydrogel network with interconnected channels and a macroporous structure was stable and achieved high cell viability (over 90%). By altering the mole ratio of sodium citrate/sodium alginate, the degradation time of the bioprinting constructs can be controlled. Cell proliferation and specific marker protein expression results also revealed that with the help of sodium citrate degradation, the printed HCECs showed a higher proliferation rate and greater cytokeratin 3(CK3) expression, indicating that this newly developed method may help to improve the alginate bioink system for the application of 3D bioprinting in tissue engineering. 

Bioprinting three-dimensional cell-laden tissue constructs with controllable degradation.

Thiolated chitosan based thermo-sensitive hydrogel is a water soluble system and the existing thiol groups are beneficial for the delivery of cysteine-rich peptides. In the present study, a kind of thiolated chitosan, i.e. chitosan-4-thio-butylamidine (CS-TBA) conjugate was characterized and used to prepare CS-TBA/hydroxyapatite (HA)/beta-glycerophosphate disodium (β-GP) thermo-sensitive hydrogel. The cysteine terminated peptide 24 (P24) containing residues 73-92 of the knuckle epitope of BMP-2 (N→C: KIPKASSVPTELSAISTLYLSGGC) was synthesized and characterized. The release behavior of P24 from CS-TBA based hydrogel was investigated in vitro. The thiol groups in CS-TBA may react with thiol groups in P24, thus decreases the P24 release rate and maintains the peptide release for a longer time compared with unmodified chitosan based hydrogel. Moreover, the bioactivity of P24 is preserved during release process. These results indicate that P24 loaded CS-TBA based thermosensitive hydrogel is a potential material for minimally invasive surgery of bone repair.

In vitro BMP-2 peptide release from thiolated chitosan based hydrogel.

Tumour metastasis is an important reason for cancer death, and cancer cell migration is an important step in the process of tumour metastasis. Studying cancer cell migration is of great significance. Here, we present a novel microfluidic co-culture system and establish mild, moderate and severe cancer models by using HMEpiC and MDA-MB-231 cells to study cancer cell migration and anti-cancer drug screening. Using this device, we achieved high cell viability (over 90%) and a stable analysis of the migration ability of cancer cells. We observed that the density of the cancer cells determined the probability of the occurrence of metastatic cells and that the induction of normal cells affected the metastatic velocity of each cancer cell. We verified that the increase in the migration ability of MDA-MB-231 cells co-cultured with HMEpiC cells was relative to the increased secretion of IL-6 and that this was verified by an IL-6 inhibitor assay. This co-culture also led to decreased CK-14 secretion and morphological changes in HMEpiC cells. Finally, significant inhibition of paclitaxel and tamoxifen on cancer migration was observed. Taken together, our microfluidic device could be a useful tool for the quantitation of the migratory capability and anti-metastatic drug screening.

Microfluidic co-culture system for cancer migratory analysis and anti-metastatic drugs screening.

While Plastic Compressed (PC) collagen technique is often used to fabricate bioengineered constructs, PC collagen gels are too weak to be sutured or conveniently handled for clinical applications. To overcome this limitation, electrospun poly (lactic-co-glycolide) (PLGA) mats, which have excellent biocompatibility and mechanical properties, were combined with PC collagen to fabricate sandwich-like hybrid constructs. By laser-perforating holes with different sizes and spacings in the electrospun mats to regulate the mechanical properties and light transmittance of the hybrid constructs, we produced hybrid constructs with properties very suitable to apply in corneal tissue engineering. The maximum tensile stress of the optimal hybrid construct was 3.42 ± 0.22 MPa. The light transmittance of the hybrid construct after perforation was approximately 15-fold higher than before, and light transmittance increased gradually with increasing time. After immersing into PBS for 7 days, the transmittance of the optimal construct changed from 63 ± 2.17% to 72 ± 1.8% under 500 nm wavelength. The live/dead staining, cell proliferation assay and immunohistochemistry study of human corneal epithelial cells (HCECs) and human keratocytes (HKs) cultured on the optimal hybrid construct both demonstrated that the cells adhered, proliferated, and maintained their phenotype well on the material. In addition, after culturing for 2 weeks, the HCECs could form stratified layers. Thus, our designed construct is suitable for the construction of engineered corneal tissue.

Tissue-engineered cornea constructed with compressed collagen and laser-perforated electrospun mat.

Future sensing applications will include high-performance features, such as toxin detection, real-time monitoring of physiological events, advanced diagnostics, and connected feedback. However, such multi-functional sensors require advancements in sensitivity, specificity, and throughput with the simultaneous delivery of multiple detection in a short time. Recent advances in 3D printing and electronics have brought us closer to sensors with multiplex advantages, and additive manufacturing approaches offer a new scope for sensor fabrication. To this end, we review the recent advances in 3D-printed cutting-edge sensors. These achievements demonstrate the successful application of 3D-printing technology in sensor fabrication, and the selected studies deeply explore the potential for creating sensors with higher performance. Further development of multi-process 3D printing is expected to expand future sensor utility and availability.

The Boom in 3D-Printed Sensor Technology.

Corneal diseases constitute the second leading cause of vision loss and affect more than 10 million people globally. As there is a severe shortage of fresh donated corneas and an unknown risk of immune rejection with traditional heterografts, it is very important and urgent to construct a corneal equivalent to replace pathologic corneal tissue. Corneal tissue engineering has emerged as a practical strategy to develop corneal tissue substitutes, and the design of a scaffold with mechanical properties and transparency similar to that of natural cornea is paramount for the regeneration of corneal tissues. Nanofibrous scaffolds produced by electrospinning have high surface area-to-volume ratios and porosity that simulate the structure of protein fibers in native extra cellular matrix (ECM). The versatilities of electrospinning of polymer components, fiber structures, and functionalization have made the fabrication of nanofibrous scaffolds with suitable mechanical strength, transparency and biological properties for corneal tissue engineering feasible. In this paper, we review the recent developments of electrospun scaffolds for engineering corneal tissues, mainly including electrospun materials (single and blended polymers), fiber structures (isotropic or anisotropic), functionalization (improved mechanical properties and transparency), applications (corneal cell survival, maintenance of phenotype and formation of corneal tissue) and future development perspectives.

Electrospun Scaffolds for Corneal Tissue Engineering: A Review.

LiFePO₄ (LFP) is a promising cathode material for lithium-ion batteries. In this study, low temperature direct writing (LTDW)-based 3D printing was used to fabricate three-dimensional (3D) LFP electrodes for the first time. LFP inks were deposited into a low temperature chamber and solidified to maintain the shape and mechanical integrity of the printed features. The printed LFP electrodes were then freeze-dried to remove the solvents so that highly-porous architectures in the electrodes were obtained. LFP inks capable of freezing at low temperature was developed by adding 1,4 dioxane as a freezing agent. The rheological behavior of the prepared LFP inks was measured and appropriate compositions and ratios were selected. A LTDW machine was developed to print the electrodes. The printing parameters were optimized and the printing accuracy was characterized. Results showed that LTDW can effectively maintain the shape and mechanical integrity during the printing process. The microstructure, pore size and distribution of the printed LFP electrodes was characterized. In comparison with conventional room temperature direct ink writing process, improved pore volume and porosity can be obtained using the LTDW process. The electrochemical performance of LTDW-fabricated LFP electrodes and conventional roller-coated electrodes were conducted and compared. Results showed that the porous structure that existed in the printed electrodes can greatly improve the rate performance of LFP electrodes.

Fabrication and Characterization of 3D-Printed Highly-Porous 3D LiFePO₄ Electrodes by Low Temperature Direct Writing Process.

A biomaterial combining fluorescent property with ability to induce osteogenesis can serve as an ideal multifunctional scaffold in bone tissue engineering. However, the frequently used fluorescent agents can only serve as imaging probes. The polymer or oligomer with a conjugated system containing nitrogen atoms will fulfill these criteria. In this study, a cyclo-trimer of acetonitrile is synthesized using a facile method, which is proved to be 4-amino-2,6-dimethylpyrimidine. The cyclo-trimer of acetonitrile demonstrates strong intrinsic photoluminescence and has the potential for in vivo imaging. The cyclo-trimer of acetonitrile shows no toxicity both in vitro and in vivo. Moreover, the cyclo-trimer of acetonitrile significantly promotes the osteogenesis of SaOS-2 cells by improving alkaline phosphatase activity, collagen type I and osteocalcin expression, as well as expressions of osteoblastic genes, and enhances the matrix mineralization of rBMSCs. Thus, the cyclo-trimer of acetonitrile synthesized in present study illustrates the employment of this kind multifunctional biomaterial in bone tissue engineering and may offer great potential in biomedical applications where bioimaging and osteogenesis are both required.

A cyclo-trimer of acetonitrile combining fluorescent property with ability to induce osteogenesis and its potential as multifunctional biomaterial.

Articular cartilage (AC) has gradient features in both mechanics and histology as well as a poor regeneration ability. The repair of AC poses difficulties in both research and the clinic. In this paper, a gradient scaffold based on poly(lactic-co-glycolic acid) (PLGA)-extracellular matrix was proposed. Cartilage scaffolds with a three-layer gradient structure were fabricated by PLGA through three-dimensional printing, and the microstructure orientation and pore fabrication were made by decellularized extracellular matrix injection and directional freezing. The manufactured scaffold has a mechanical strength close to that of real cartilage. A quantitative optimization of the Young's modulus and shear modulus was achieved by material mechanics formulas, which achieved a more accurate mechanical bionic and a more stable interface performance because of the one-time molding process. At the same time, the scaffolds have a bionic and gradient microstructure orientation and pore size, and the stratification ratio can be quantitatively optimized by design of the freeze box and temperature simulation. In general, this paper provides a method to optimize AC scaffolds by both mechanics and histology as well as a bionic multimaterial scaffold. This paper is of significance for cell culture and clinical transplantation experiments. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1664-1676, 2018.

Construction of bionic tissue engineering cartilage scaffold based on three-dimensional printing and oriented frozen technology.

The liver is one of the main metabolic organs, and nearly all ingested drugs will be metabolized by the liver. Only a small fraction of drugs are able to come onto the market during drug development, and hepatic toxicity is a major cause for drug failure. Since drug development is costly in both time and materials, an in vitro liver model that can accelerate bioreactions in the liver and reduce drug consumption is imperative in the pharmaceutical industry. The liver on a chip is an ideal alternative for its controllable environment and tiny size, which means constructing a more biomimetic model, reducing material consumption as well as promoting drug diffusion and reaction. In this study, taking advantage of the laminar flow on chips and using natural degradable gel rat tail Collagen-I, we constructed a liver sinusoid on a chip. By synchronously injecting two kinds of cell-laden collagen, HepG2-laden collagen and HUVEC-laden collagen, we formed two collagen layers with a clear borderline. By controlling the HUVEC density and injection of growth factors, HUVECs in collagen formed a monolayer through self-assembly. Thus, a liver sinusoid on a chip was achieved in a more biomimetic environment with a more controllable and uniform distribution of discrete HUVECs. Viability, album secretion and urea synthesis of the live sinusoid on a chip were analysed on days 3, 5 and 7 after collagen injection with acetaminophen treatment at 0 (control), 10 and 20 mM. The results indicated that our liver sinusoid on a chip was able to maintain bioactivity and function for at least 7 d and was beneficial for hepatotoxic drug screening.

Construction of a liver sinusoid based on the laminar flow on chip and self-assembly of endothelial cells.

Corneal diseases are the main reason of vision loss globally. Constructing a corneal equivalent which has a similar strength and transparency with the native cornea, seems to be a feasible way to solve the shortage of donated cornea. Electrospun collagen scaffolds are often fabricated and used as a tissue-engineered cornea, but the main drawback of poor mechanical properties make it unable to meet the requirement for surgery suture, which limits its clinical applications to a large extent. Aligned polyvinyl acetate (PVA)/collagen (PVA-COL) scaffolds were electrospun by mixing collagen and PVA to reinforce the mechanical strength of the collagen electrospun scaffold. Human keratocytes (HKs) and human corneal epithelial cells (HCECs) inoculated on aligned and random PVA-COL electrospun scaffolds adhered and proliferated well, and the aligned nanofibers induced orderly HK growth, indicating that the designed PVA-COL composite nanofibrous electrospun scaffold is suitable for application in tissue-engineered cornea.

Engineering of Corneal Tissue through an Aligned PVA/Collagen Composite Nanofibrous Electrospun Scaffold.

Tumour invasion into the surrounding stroma is a critical step in metastasis, and it is necessary to clarify the role of microenvironmental factors in tumour invasion. We present a microfluidic system that simulated and controlled multi-factors of the tumour microenvironment for three-dimensional (3D) assessment of tumour invasion into the stroma. The simultaneous, precise and continuous arrangement of two 3D matrices was visualised to observe the migration of cancer cell populations or single cells by transfecting cells with a fluorescent protein. A vascular endothelial layer was formed to simulate transendothelial transport of nutrients, and its endothelial barrier function was verified by the diffusion of 70 kDa fluorescein isothiocyanate (FITC)-Dextran in 3D matrices. Through high-throughput cell migration tracking observation and statistic evaluation, we clarified that cell density of the tumour directly determined its invasiveness. The results suggested that increased secretion of IL-6 among both cancer cells (MDA-MB-231) and noncancerous cells (MCF-10A or HDF-n) after co-culture contributes to cancer cell invasiveness, and this was verified by an IL-6 inhibitor assay. Finally, the drug efficacy of paclitaxel was reflected as changes in cancer cell migration ability, viability, and morphology. Together, our microfluidic devices could be a useful tool to study the mechanism of tumour invasion into the stroma and to screen anti-metastatic drugs.

Shengli Mi

Graduate School at Shenzhen

Tsinghua University

Generation of Size-controlled Poly (ethylene Glycol) Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices