Bilayered Construct Research Articles

A bioprinted vascularized skin substitute with fibroblasts, keratinocytes, and endothelial progenitor cells for skin wound healing

Skin substitutes are highly demanded by patients with extensive burns and full-thickness skin wounds. Bioprinting offers a promising technology to fabricate customized cell-laden skin substitutes. In this study, a hydrogel of biodegradable polyurethane (PU)-gelatin (4:1) laden with human fibroblasts, endothelial progenitor cells (EPCs), and keratinocytes was used as the bioink for building a bi-layer dermo-epidermal skin substitute. The seven-layer cell-laden constructs with stack thickness of 1.4 mm were precisely deposited through a 210 μm nozzle with an air pressure of 0.055–0.175 MPa and nozzle temperature of 19 °C. When grown in vitro, three types of cells in the bioprinted constructs showed good cell viability (>99%) in 24 h and reached a high proliferation rate (>220%) in 14 days. Significantly, EPCs were successfully differentiated into endothelial-like cells in the constructs and expressed the vasculogenesis-related proteins (CD31+ and eNOS+). When implanted in vivo, the bi-layer constructs attained ∼90% wound healing ratio and ∼76% re-epithelialization after 28 days in the nude mice model. Histological analyses revealed that skin wounds treated with the bi-layer constructs achieved high degrees of tissue integration and collagen production after 28 days. Vasculogenesis and angiogenesis of the wound treated with bi-layer constructs was significantly greater (∼300%) than those of the untreated wounds. The in vitro and in vivo findings indicate that the bioprinted skin substitutes with fibroblasts, EPCs, and keratinocytes embedded in PU-gelatin hydrogel may offer a promising strategy for clinical wound treatment and development of bioprinted skin.

492 Bi-layered skin construct with gene modified keratinocytes reduces UVR DNA damage

Ultraviolet radiation (UVR) is among major contributors to skin cancer in humans. Production of melanin serves as a main defense mechanisms against UVR damage. This pigment, which gives color to hair, skin and eyes, protects the underlying tissue from UVR damage. However, in some patient populations, this protection mechanism is not effective, leaving patients highly susceptible to cancer. Similarly, mycosporine like amino acids (MAAs), which are commonly expressed in marine algae, have been shown to serve a protective role in UVR-induced DNA damage.

Theoretical stiffness limits of 4D printed self-folding metamaterials

4D printing of flat sheets that self-fold into architected 3D structures is a powerful origami-inspired approach for the fabrication of multi-functional devices and metamaterials. However, the opposite stiffness requirements for the folding process and the subsequent loadbearing of 3D structures impose an intrinsic limitation in designing self-folding metamaterials: while a low stiffness is required for the successful completion of the self-folding step, a high stiffness is needed for utilizing the folded structure as a load-bearing mechanical metamaterial. Here, we present a nonlinear analytical model of self-folding bilayer constructs composed of an active and passive layer. This finite-deformation theoretical model predicts the curvature of activated bilayers, establishes their stability limits, and estimates the stiffness of folded bilayers, yielding the theoretical stiffness limits of self-folding bilayers. We use our model to identify the optimal combinations of geometrical and mechanical properties that result in the highest possible stiffness of folded constructs. We then compare the predictions of our analytical model with computational results, and validate our theory with experimental realizations of 4D printed structures. Finally, we evaluate the theoretical stiffness limits of bilayer constructs made using the most common types of stimuli-responsive materials. Our analysis shows that a maximum effective modulus of ≈ 1.5 GPa can be achieved using the currently available shape-memory polymers.

89 Pooled Safety Analysis Evaluating Bioengineered Allogeneic Cellularized Construct in Patients with Deep Partial-thickness Thermal Burns

IntroductionAutograft (AG) is the standard of care treatment for deep burns but requires creation of a donor site wound prone to pain and scarring. Treatment with a bioengineered allogeneic cellularized construct (BACC) is an alternative approach that can reduce or eliminate the need for autografting. The BACC is a bilayer construct that was recently approved in the US for the treatment of adult deep partial-thickness (DPT) burns. Here, we report the analysis of pooled safety data from two open-label, randomized, controlled trials (STRATA2011 [NCT01437852] and STRATA2016 [NCT03005106]) that evaluated efficacy and safety of BACC versus autografting in patients with DPT burns.MethodsThe trials enrolled 101 patients aged ≥18 years with 3–49% total body surface area (TBSA) thermal burns. In each patient, two DPT areas on the torso or extremities were randomized to receive BACC or AG, where the mean total BACC dosage was 234.8 cm2 (range: 12.0–960.0 cm2). The safety endpoints assessed at each visit included: 1) treatment-emergent adverse events (TEAEs), treatment-related AEs (TRAEs), and serious AEs (SAEs); 2) changes in immunologic responses (panel reactive antibodies [PRA], anti-bovine serum albumin [BSA] antibody response [STRATA2016 only]); 3) persistence of allogeneic DNA; and 4) laboratory exam and vital signs.ResultsEighty-seven patients (86.1%) experienced TEAEs, 30 patients (29.7%) experienced TRAEs, and 16 patients (15.8%) experienced SAEs. The most frequent TEAEs reported by ≥10% of patients in the pooled analysis were pruritus (n=31, 30.7%) and blister, hypertension, and hypertrophic scar (n=11, 10.9% each). The most frequent TRAEs (≥5% of patients) were pruritus (n=13, 12.9%) and blister (n=5, 5%). The most common SAEs were transplant (BACC or AG) failure, pneumonia, and deep vein thrombosis (n=2, 2% each), where only one SAE (impaired healing of moderate severity) was possibly related to BACC. One patient (1%) discontinued the trial due to a TEAE (traumatic brain injury). Two patients (2%) experienced SAEs that led to death, neither related to BACC. The number of patients with positive PRA values that were negative at baseline were 36 (38.7%) at Day 28 and 20 (22%) at Month 3. The number of patients with reactivity to HLA I class alleles found in the BACC increased from 4 (4%) at baseline to 39 (40.9%) at Day 28, then decreased to 22 (24.2%) at Month 3. No persistence of allogeneic DNA from the BACC was detected.ConclusionsBACC is well tolerated and is not associated with any unexpected SAEs or TEAEs. The safety profile at BACC treatment sites is similar to that at AG treatment sites. Thus, BACC may offer a safe alternative to autografting for the treatment of DPT burns.

812 Evaluation of Dermal and Epidermal Replacement Strategies for the Treatment of Full-thickness Wounds

IntroductionIn full-thickness (FT) wounds, autologous skin cell suspension (ASCS) is used in combination with widely meshed split-thickness skin grafts (mSTSGs) to obtain definitive closure, reducing donor skin requirements compared to traditional autografting (AG) techniques. In the treatment of these wounds, dermal matrices (DMs) are also often utilized to address various challenges including need for temporization, mitigation of contour defects, covering avascular structures, and modulation of scar formation. Varying DMs exist and are composed of different biologic and synthetic materials designed to address these clinical needs. The purpose of this study was to compare outcomes obtained in FT wounds using 3 different DMs coupled with ASCS+mSTSG in immediate or delayed AG procedures.MethodsA FT excisional porcine wound model was used. DMs evaluated included a single-layer dermal matrix composed of bovine dermal collagen and elastin (Col/E), a bilayer construct composed of a bovine collagen-glycosaminoglycan with a silicone epidermal layer (Col/GAG), and a synthetic bilayer polyurethane DM (Poly/U).DMs were applied, managed, and grafted following manufacturer’s recommendations. AG included ASCS+mSTSG (1:80 expansion, 3:1 mesh). Wounds were evaluated for inflammation, infection, DM take, AG take, re-epithelialization, and contracture over 49 days post-excision. Additionally, biopsies were evaluated to further inform tissue generation and healing outcomes.ResultsResults related to healing outcomes are reported in the table below. Inflammation at wound margins was noted during acute phase of healing for all DMs and visual cues for infection were noted in 1 wound for Col/E, 3 wounds for Col/GAG causing partial loss of DM, and 1 wound for Poly/U. All signs of infection were resolved by day 14.ConclusionsASCS+mSTSG can be used successfully over DMs composed of various materials in both immediate and delayed AG procedures. No difference was observed on percent AG take between the DMs, however the data suggest that time to definitive closure is impacted based on utilized DM and AG strategy, with potential implications on contracture.

Quasi‐2D Bilayer Surface Passivation for High Efficiency Narrow Bandgap Perovskite Solar Cells

AbstractThe combination of comprehensive surface passivation and effective interface carriers transfer plays a critical role in high‐performance perovskite solar cells. A 2D structure is an important approach for surface passivation of perovskite film, however, its large band gap could compromise carrier transfer. Herein, we synthesize a new molecule 2‐thiopheneethylamine thiocyanate (TEASCN) for the construction of bilayer quasi‐2D structure precisely on a tin‐lead mixed perovskite surface. This bilayer structure can passivate the perovskite surface and ensure effective carriers transfer simultaneously. As a result, the open‐circuit voltage (Voc) of the device is increased without sacrificing short‐circuit current density (Jsc), giving rise to a high certified efficiency from a credible third‐party certification of narrow band gap perovskite solar cells. Furthermore, theoretical simulation indicates that the inclusion of TEASCN makes the bilayer structure thermodynamically more stable, which provides a strategy to tailor the number of layers of quasi‐2D perovskite structures.

An autonomously swimming biohybrid fish designed with human cardiac biophysics.

Biohybrid systems have been developed to better understand the design principles and coordination mechanisms of biological systems. We consider whether two functional regulatory features of the heart-mechanoelectrical signaling and automaticity-could be transferred to a synthetic analog of another fluid transport system: a swimming fish. By leveraging cardiac mechanoelectrical signaling, we recreated reciprocal contraction and relaxation in a muscular bilayer construct where each contraction occurs automatically as a response to the stretching of an antagonistic muscle pair. Further, to entrain this closed-loop actuation cycle, we engineered an electrically autonomous pacing node, which enhanced spontaneous contraction. The biohybrid fish equipped with intrinsic control strategies demonstrated self-sustained body-caudal fin swimming, highlighting the role of feedback mechanisms in muscular pumps such as the heart and muscles.

An electrostatic finite element analysis of the electrospinning process of bilayer constructs using a parallel-plate collector

Fundamental knowledge of the deposition process of electrospun fibers is of paramount importance to the fabrication of constructs with fibers oriented in specific directions, depending on the mandrel geometry. This paper presents an electrostatic finite element analysis of a parallel-plate electrospinning configuration that reveals the effects of the electric potential intensity and directionality on the deposition of aligned and randomly oriented fibers, resulting in the formation of a fibrous bilayer construct. Simulations demonstrate that polymer deposition between the collector plates yields a local effect on the electric field. This study provides a computational methodology for investigating other collector configurations and guidance for designing electrospinning setups capable of producing microfibrous constructs with tailored morphologies.

Effect of Clothing Fabric on 20-km Cycling Performance in Endurance Athletes.

Purpose: Examine the effect of synthetic fabrics (SYN, 60% polyester: 40% nylon) vs. 100% cotton fabric (CTN) on the 20-km cycling time trial (20 kmCTT) performance of competitive cyclists and triathletes.Methods: In this randomized controlled crossover study, 15 adults (5 women) aged 29.6 ± 2.7 years (mean ± SE) with a peak rate of O2 consumption of 60.0 ± 2.0 ml/kg/min completed a 20 kmCTT under ambient laboratory conditions (24.3 ± 0.7°C and 17 ± 7% relative humidity) with a simulated wind of ~3 m/s while wearing SYN or CTN clothing ensembles. Both ensembles were of snowflake mesh bi-layer construction and consisted of a loose-fitting long-sleeved shirt with full-length trousers.Results: Participants maintained a significantly (p < 0.05) higher cycling speed and power output over the last 6-km of the 20 kmCTT while wearing the SYN vs. CTN ensemble (e.g., by 0.98 km/h and 18.4 watts at the 20-km mark). Consequently, 20 kmCTT duration was significantly reduced by 15.7 ± 6.8 sec or 0.8 ± 0.3% during SYN vs. CTN trials (p < 0.05). Improved 20 kmCTT performance with SYN vs. CTN clothing could not be explained by concurrent differences in esophageal temperature, sweat rate, ratings of perceived exertion and/or cardiometabolic responses to exercise. However, it was accompanied by significantly lower mean skin temperatures (~1°C) and more favorable ratings of perceived clothing comfort and thermal sensation during exercise.Conclusion: Under the experimental conditions of the current study, athletic clothing made of synthetic fabrics significantly improved the 20 kmCTT performance of endurance-trained athletes by optimizing selected thermoregulatory and perceptual responses to exercise.

High-performance bilayer composites for the replacement of osteochondral defects.

Osteochondral (OC) defects combine damage to cartilage and subchondral bone, posing a significant challenge to their repair due to the dissimilar characteristics and regenerative capabilities between the two tissues. Here, we propose novel OC bilayer composites, drawing inspiration from corresponding biological tissues and using a combination of simple and reproducible techniques. Cartilage-like materials based on poly(vinyl alcohol) (PVA) hydrogels were produced with nanofiber reinforcements acquired from high-performance fibers (Kevlar® and Zylon®), while bone-like materials were obtained by adding magnesium-substituted calcium phosphate ceramics to PVA. All composites were sterilized by gamma irradiation to rule out the possibility of undesirable effects resulting from the process, and then fully characterized. The results indicated that nanofibers and bioceramics incorporated into the PVA networks form promising structures with multiple interesting properties. The composites resembling cartilage and bone showed high biomimicry with natural tissues, being able to reconcile exceptional mechanics with the requirements of adequate porosity, liquid content, and biological behavior. The developed materials reveal a high potential for use in OC tissue repair applications.

Functionalized Fluoropolymer-Compatibilized Elastomeric Bilayer Composites for Osteochondral Repair: Unraveling the Role of Substrate Stiffness and Functionalities

The osteochondral lesions and osteoarthritis-related complications continue to be clinically relevant challenges to be addressed by the biomaterials community. Hydrogel-based scaffolds have been widely investigated to enhance osteochondral regeneration, but the inferior mechanical properties together with poor functional stability are the major constraints in their clinical translation. The development of osteochondral implants with natural tissue-mimicking mechanical properties remains largely unexplored. In this perspective, the present study demonstrates a strategy to develop a bilayer osteochondral implant with an elastically stiff composite (poly(vinylidene difluoride)-reinforced BaTiO3, PVDF/BT) and elastically compliant composite (maleic anhydride-functionalized PVDF/thermoplastic polyurethane/BaTiO3, m-PVDF/TPU/BT). The compositional variation in polymer composites allowed the elastic modulus of the hybrid bilayer construct to vary from ∼2 GPa to ∼90 MPa, which enabled a better understanding of the substrate-stiffness-dependent cellular behavior and maturation of preosteoblasts and chondrocytes. The cellular functionalities on PVDF-based polymer matrices have been benchmarked against ultrahigh-molecular-weight polyethylene (UHMWPE), which is clinically used for a wide spectrum of orthopedic applications. The increased alkaline phosphatase (ALP) activity, collagen synthesis, and matrix mineralization confirmed the early differentiation of preosteoblasts on the PVDF/BT matrix with subchondral bone-like mechanical properties. On the contrary, the upregulated chondrogenic functionalities were recorded on m-PVDF/TPU/BT with an elevated level of collagen content, glycosaminoglycans, and proteoglycans. Emphasis has been laid on probing the regulation of the osteochondral behavior using tailored substrate stiffness and functionalities using compatibilized fluoropolymer-based elastomeric composites. Taken together, the results of this work conclusively establish the efficacy of the hybrid bilayer composite with natural tissue-mimicking mechanical properties for the functional repair of osteochondral defects.

Silk-based bilayered small diameter woven vascular conduits for improved mechanical and cellular characteristics

A major impediment to the development of small diameter vascular grafts is to achieve an optimal balance between its mechanical properties and cellular response. To address this, the technique of cylindrical weaving has been combined with electrospinning to fabricate a seamless bilayered conduit (∼3 mm) having an inner cell-friendly nanofibrous layer of poly(caprolactone)/collagen and an outer mechanically compliant woven silk layer. Mechanical characteristics such as burst strength, suture retention, compliance and leak resistance were found to be improved for this bilayered conduit when compared to the commercial standard. In-vitro studies revealed that the lumen of the conduit was non-hemolytic and could support adhesion and viability of endothelial cells. Overall, our studies suggest that the proposed bilayer construct could be a suitable candidate for small diameter vascular prosthesis.

Hydration-induced reversible deformation of the pine cone

The scales of pine cones undergo reversible deformation due to hydration changes in order to optimize seed dispersal. This improves the survivability of the pine. The reversible flexing of the scales is caused by two tissue layers arranged in a sandwich configuration: a layer composed of sclereid cells and a sclerenchyma layer. They expand differentially upon hydration (and contract upon dehydration) due to differences in the structure that are analyzed here for Torrey pine (Pinustorreyana) cones. In addition to this well-known mechanism by which the cellulose microfibrils in the scales vary their angle with the wood cell axis, we confirm the presence of a porosity gradient in the sclereid cells and calculate, using a model consisting of three layers, the stresses generated upon dehydration taking into account the effect of hydration on the elastic modulus. Our quantitative analysis reveals that this gradient structure can significantly decrease the stress concentrations due to the mismatch between the two layers, and show that this is an ingenious design to increase the interfacial toughness to improve the robustness of pine cone scales. We also show that each individual layer of sclereid cells and sclerenchyma fibers undergoes bending when hydrated separately, and suggest that the two layers operate synergistically to effect the required deformation for seed release. A synthetic bioinspired analog consisting of hydrogels with different porosities is used to confirm this principal actuation mechanism. These findings may inspire the materials science and mechanical engineering communities to develop more robust, biocompatible and energy-efficient actuation systems. Statement of significanceSome biological structures can exhibit reversible deformation enabled by water inflow and outflow of their structure. We analyse the reversible motion of pine cone scales. The dehydration produces their flexure and opening, resulting in the release of seeds and their dispersal, when conditions are right. This process is reversible, and rehydration of the pine cone recloses the scales. The processes of flexing and straightening are governed by shrinking and swelling which are directed by differences in the arrangement of cellulose microfibrils in a bilayer construct. We demonstrate that the scales are more complex than a simple bilayer structure and that they actually have gradients, which significantly reduce the internal stresses and ensure their integrity. We analyse the process of opening and closing of the scales for a gradient structure in the Torrey pine cone using a simple idealized trilayer model. The results demonstrate a significant decrease in internal stresses produced by the gradient structure. Using the lessons learned from the pine cone, we produce a bilayer junction using hydrogels with different porosities which exhibit the same reversible bending response.

Nanoengineered highly sensitive and stable soft strain sensor built from cracked carbon nanotube network/composite bilayers

Sensitivity and stretchability are two key physical parameters for advanced piezoresistive sensors, especially for those that can be incorporated into clothing or attached directly on human body. Big challenge remains in achieving both in single device. Here, to obtain high strain sensitivity and wide measurement range simultaneously, a nano-engineered bilayer composed of cracked carbon nanotube (CNT) network and elastomer infiltrated CNT composite has been developed. This design takes advantage of the cracked CNT network layer which is able to provide the sensor with high resistance sensitivity (gauge factor between 8 and 207), wide strain measurement range (>50%) and good linearity (R2 > 93%) in three strain ranges. The coupled elastomer nanocomposite layer is utilized to confine the crack evolution resulting in significantly improved stability and reproducibility (>10,000 loading-unloading cycles). Application of such bilayer construction also processes fast response time (<10 ms) and cyclic drift less than 0.01% in human motion detection. Piezoresistive responses of sensors can be designed and effectively tuned to meet specific requirements through the choosing of CNT sponge properties and control of individual layer thickness.

Hydration-induced reversible deformation of biological materials

The influx and efflux of water in biological structures actuates reversible deformation and recovery processes that are crucial for mechanical functions in plants and animals. These processes utilize various mechanochemical mechanisms: swelling directed by the arrangement of cellulosic microfibrils in a bilayer construct, which generates different deformation patterns; lignification gradients; hierarchical foam-like inner structures, some of which also include swelling by hygroscopic cellulose inner cell layer; turgor pressure, which is activated by osmosis and acts at the cellular level, generating reversible motions. In this Review, we present representatives of each of these four mechanisms: pine cones, wheat awns, the twisted opening of Bauhinia pods and the seed of the stork’s bill; the resurrection plant; ice plant seed capsules and carrotwood seed pod; the wilting and redressing of plant stems. Natural polymeric materials produced by animals can also exhibit hydration-driven shape and strength recovery: bird feathers and hair are prime examples. Spider silk — a non-keratinous biopolymer — also exhibits humidity-driven reversible deformation. After describing these animal-based mechanisms, we outline bioinspired applications to actuate multifunctional and biocompatible smart materials, and indicate future directions of research with potential for new bioinspired designs. The influx and efflux of water in biological structures leads to reversible deformation, which has important functions in plants (for example, in seed protection and dispersal) and animals (for example, in the recovery of the strength and shape of feathers, and for reversible changes in silk and hair). Here the authors review the main hydration-induced deformation mechanisms and highlight applications inspired by these processes.

Interface-by-design in zirconia-polyurea matrix hybrid composites

This study investigates the incorporation of zirconia particles in polyurea matrix to form hybrid composites. Pristine zirconia particles in the monoclinic phase are first premixed with an oligomeric diamine−a reactant for polyurea−as an alternative method of surface functionalization that does not require the use of organosilanes. Interfacial interactions between the zirconia particles and polyurea are revealed via attenuated total reflectance-Fourier transform infrared spectroscopy, where a shift and broadening of the urea-carbonyl stretching and an increase in relative intensity of the corresponding disordered vs. ordered N–H stretching are shown in hybrid composites vs. bulk polyurea. The composition dependence of the viscoelastic relaxation characteristics and dynamic storage modulus in hybrid composites as determined by dynamic mechanical analysis are also elaborated upon. Additionally, we evaluate the potential of exploiting zirconia-polyurea hybrids to enabling dynamic impedance optimization at the interface of a select bi-layer construct, particularly when considering the material response under dynamic loading environments.

Establishing multiple osteogenic differentiation pathways of mesenchymal stem cells through different scaffold configurations

Mimicking the complex organization of the extracellular matrix (ECM), especially its structure and dimensionality, is necessary to produce living tissues from stem cells. In compliance with a previously established role of nanofiber organization for the osteogenic differentiation of stem cells, here we used hybrid fibrinogen/poly(l-lactide-ε-caprolactone) (FBG/PLCL) nanofibers arranged in aligned and honeycomb configurations, to recapitulate the highly oriented ECM of the cortical bone and the sponge-like (i.e., honeycomb) environment of the cancellous one, respectively. Using special bilayered constructs, we demonstrate that the dimensionality (i.e., 2D vs. 3D) of the nanofibers as well as their architecture (i.e., honeycomb vs. aligned) affects differently the overall morphology and the expression of multiple osteogenic genes of human adipose-derived mesenchymal stem cells (ADMSCs). The cells had elongated shape with markedly increased cell mobility when seeded on aligned nanofibers. Conversely, on honeycomb-shaped nanofibers, ADMSCs initially concentrated inside the honeycomb curvatures adopting rounded morphology, but late, they formed network-like structures overlaying the honeycomb curvatures. By employing quantitative polymerase chain reaction (qPCR), we further show that a 3D environment generally supports the multiple osteogenic response of ADMSCs, but honeycomb and aligned architectures promote rather different differentiation pathways.

Repair of Osteochondral Defects With Predifferentiated Mesenchymal Stem Cells of Distinct Phenotypic Character Derived From a Nanotopographic Platform

Background: Articular cartilage has a zonal architecture and biphasic mechanical properties. The recapitulation of surface lubrication properties with high compressibility of the deeper layers of articular cartilage during regeneration is essential in achieving long-term cartilage integrity. Current clinical approaches for cartilage repair, especially with the use of mesenchymal stem cells (MSCs), have yet to restore the hierarchically organized architecture of articular cartilage. Hypothesis: MSCs predifferentiated on surfaces with specific nanotopographic patterns can provide phenotypically stable and defined chondrogenic cells and, when delivered as a bilayered stratified construct at the cartilage defect site, will facilitate the formation of functionally superior cartilage tissue in vivo. Study Design: Controlled laboratory study. Methods: MSCs were subjected to chondrogenic differentiation on specific nanopatterned surfaces. The phenotype of the differentiated cells was assessed by the expression of cartilage markers. The ability of the 2-dimensional nanopattern-generated chondrogenic cells to retain their phenotypic characteristics after removal from the patterned surface was tested by subjecting the enzymatically harvested cells to 3-dimensional fibrin hydrogel culture. The in vivo efficacy in cartilage repair was demonstrated in an osteochondral rabbit defect model. Repair by bilayered construct with specific nanopattern predifferentiated cells was compared with implantation with cell-free fibrin hydrogel, undifferentiated MSCs, and mixed-phenotype nanopattern predifferentiated MSCs. Cartilage repair was evaluated at 12 weeks after implantation. Results: Three weeks of predifferentiation on 2-dimensional nanotopographic patterns was able to generate phenotypically stable chondrogenic cells. Implantation of nanopatterned differentiated MSCs as stratified bilayered hydrogel constructs improved the repair quality of cartilage defects, as indicated by histological scoring, mechanical properties, and polarized microscopy analysis. Conclusion: Our results indicate that with an appropriate period of differentiation, 2-dimensional nanotopographic patterns can be employed to generate phenotypically stable chondrogenic cells, which, when implanted as stratified bilayered hydrogel constructs, were able to form functionally superior cartilage tissue. Clinical Relevance: Our approach provides a relatively straightforward method of obtaining large quantities of zone-specific chondrocytes from MSCs to engineer a stratified cartilage construct that could recapitulate the zonal architecture of hyaline cartilage, and it represents a significant improvement in current MSC-based cartilage regeneration.

Direct-Write Bioprinting Approach to Construct Multilayer Cellular Tissues.

As a cellular-assembly technique, bioprinting has been extensively used in tissue engineering and regenerative medicine to construct hydrogel-based three-dimensional (3D) tissue-like models with prescribed geometry. Here, we introduced a unique direct-write bioprinting strategy to fabricate a bilayer flat tissue in a hydrogel-free approach. A printed retina pigmented epithelium layer (RPE) was applied as living biopaper for positioning a fibroblast layer without using any hydrogel in bioink. We adjusted the number of cells in the inkjet droplets in order to obtain a uniform printed cell layer and demonstrated the formation of a bilayer construct through confocal imaging. Since our printing system introduced low levels of shear stress to the cells, it did not have a negative effect on cell survival, although cell viability was generally lower than that of control group over 1 week post-printing. In conclusion, our novel direct-write bioprinting approach to spatiotemporally position different cellular layers may represent an efficient tool to develop living constructs especially for regeneration of complex flat tissues.

Quantitative Analysis of Cold Stress Inducing Lipidomic Changes in Shewanella putrefaciens Using UHPLC-ESI-MS/MS.

Shewanella putrefaciens is a well-known specific spoilage organism (SSO) and cold-tolerant microorganism in refrigerated fresh marine fish. Cold-adapted mechanism includes increased fluidity of lipid membranes by the ability to finely adjust lipids composition. In the present study, the lipid profile of S. putrefaciens cultivated at 30, 20, 10, 4, and 0 °C was explored using ultra-high-pressure liquid chromatography/electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) to discuss the effect of lipid composition on cold-adapted tolerance. Lipidomic analysis detected a total of 27 lipid classes and 606 lipid molecular species in S. putrefaciens cultivated at 30, 20, 10, 4, and 0 °C. S. putrefaciens cultivated at 30 °C (SP-30) had significantly higher content of glycerolipids, sphingolipids, saccharolipids, and fatty acids compared with that at 0 °C (SP-0); however, the lower content of phospholipids (13.97%) was also found in SP-30. PE (30:0), PE (15:0/15:0), PE (31:0), PA (33:1), PE (32:1), PE (33:1), PE (25:0), PC (22:0), PE (29:0), PE (34:1), dMePE (15:0/16:1), PE (31:1), dMePE (15:1/15:0), PG (34:2), and PC (11:0/11:0) were identified as the most abundant lipid molecular species in S. putrefaciens cultivated at 30, 20, 10, 4, and 0 °C. The increase of PG content contributes to the construction of membrane lipid bilayer and successfully maintains membrane integrity under cold stress. S. putrefaciens cultivated at low temperature significantly increased the total unsaturated liquid contents but decreased the content of saturated liquid contents.