Multilayered Scaffolds Research Articles

Cell-free osteochondral scaffolds provide a substantial clinical benefit in the treatment of osteochondral defects at a minimum follow-up of 5\u2009years

PurposeThe treatment of osteochondral lesions is challenging and no consensus has been established about the best option for restoring both cartilage and subchondral bone. Multilayer collagen-hydroxyapatite scaffolds have shown promising clinical results, but the outcome at a follow-up longer than 5 years still has to be proved. The aim was to evaluate the clinical outcome of patients with a knee isolated osteochondral lesion treated with a biomimetic three-layered scaffold at a minimum 5 years of follow-up.MethodsTwenty-nine patients (23 males and 6 females, mean age 31.5 ± 11.4 years) were evaluated retrospectively before surgery, at 1 and 2 years and at last follow-up (FU). Visual Analog Scale (VAS) for pain, International Knee Documentation Committee (IKDC) Subjective Score, Tegner-Lysholm Knee Scoring Scale and Tegner Activity Level Scale were collected. Mean FU was 7.8 ± 2.0 years (min 5.1 - max 11.3). The etiology of the defect was Osteochondritis Dissecans or osteonecrosis (17 vs 12 cases).ResultsAt 12 months FU the IKDC score improved from 51.1 ± 21.7 to 80.1 ± 17.9 (p < 0.01), Tegner Lysholm Score from 59.9 ± 17.3 to 92.5 ± 9.0 (p < 0.01), VAS from 6.1 ± 2.1 to 1.7 ± 2.3 (p < 0.01) and Tegner Activity Level Scale from 1.6 ± 0.5 to 4.9 ± 1.7 (p < 0.01). The results remained stable at 24 months, while at last FU a statistically significant decrease in IKDC, Tegner Lysholm and Tegner Activity Scale was recorded, though not clinically relevant. Patients under 35 achieved statistically better outcomes.ConclusionsThe use of a cell-free collagen-hydroxyapatite osteochondral scaffold provides substantial clinical benefits in the treatment of knee osteochondral lesions at a minimum follow-up of 5 years, especially in patients younger than 35 years.Level of evidenceLevel IV.

Development of a Multi-Layer Skin Substitute Using Human Hair Keratinic Extract-Based Hybrid 3D Printing.

Large-sized or deep skin wounds require skin substitutes for proper healing without scar formation. Therefore, multi-layered skin substitutes that mimic the genuine skin anatomy of multiple layers have attracted attention as suitable skin substitutes. In this study, a novel skin substitute was developed by combining the multi-layer skin tissue reconstruction method with the combination of a human-derived keratinic extract-loaded nano- and micro-fiber using electrospinning and a support structure using 3D printing. A polycaprolactone PCL/keratin electrospun scaffold showed better cell adhesion and proliferation than the keratin-free PCL scaffold, and keratinocytes and fibroblasts showed better survival, adhesion, and proliferation in the PCL/keratin electrospun nanofiber scaffold and microfiber scaffold, respectively. In a co-culture of keratinocytes and fibroblasts using a multi-layered scaffold, the two cells formed the epidermis and dermal layer on the PCL/keratin scaffold without territorial invasion. In the animal study, the PCL/keratin scaffold caused a faster regeneration of new skin without scar formation compared to the PCL scaffold. Our study showed that PCL/keratin scaffolds co-cultured with keratinocytes and fibroblasts promoted the regeneration of the epidermal and dermal layers in deep skin defects. Such finding suggests a new possibility for artificial skin production using multiple cells.

Fabrication and in vitro evaluation of PCL/gelatin hierarchical scaffolds based on melt electrospinning writing and solution electrospinning for bone regeneration

As an emerging 3D printing technique, melt electrospinning writing (MEW) has been used to fabricate scaffolds with controllable structure and good mechanical strength for bone regeneration. However, how to further improve MEW scaffolds with nanoscale extracellular matrix (ECM) mimic structure and bioactivity is still challenging. In this study, we proposed a simple composite process by combining MEW and solution electrospinning (SE) to fabricate a micro/nano hierarchical scaffold for bone tissue engineering. The morphological results confirmed the hierarchical structure with both well-defined MEW microfibrous grid structure and SE random nanofiber morphology. The addition of gelatin nanofibers turned the scaffolds to be hydrophilic, and led to a slight enhancement of mechanical strength. Compared with PCL MEW scaffolds, higher cell adhesion efficiency, improved cell proliferation and higher osteoinductive ability were achieved for the MEW/SE composite scaffolds. Finally, multilayer composite scaffolds were fabricated by alternately stacking of MEW layer and SE layer and used to assess the effect on cell ingrowth in the scaffolds. The results showed that gelatin nanofibers did not inhibit cell penetration, but promoted the three-dimensional growth of bone cells. Thus, the strategy of the combined use of MEW and SE is a potential method to fabricate micro/nano hierarchical scaffolds to improve bone regeneration.

Biofunctionalized Nanofibrous Bilayer Scaffolds for Enhancing Cell Adhesion, Proliferation and Osteogenesis.

A simple and efficient method for fabricating functionalized multilayered nanofibrous scaffolds has been developed by combining electrospinning and thermally induced phase separation (TIPS) techniques. In this investigation, functionalized bilayer scaffolds were constructed using this method for bone tissue engineering, which consisted of a nanofibrous poly(lactic acid-co-glycolic acid) (PLGA) membrane as the base and a nanofibrous chitosan (CS) or gelatin (Gel) mesh as the surface layer, with the PLGA nanofibers having a biomimetic polydopamine (PDA) coating. It was shown that the PDA coating strongly bonded TIPS-formed CS or Gel networks onto PDA-coated electrospun PLGA membranes. The nanofibrous PLGA membrane provided adequate mechanical support for the whole structure, and the nanofibrous CS or Gel networks enhanced cell growth and maturation. The bioinspired surface modification of PLGA scaffolds through PDA coating could not only provide strong adhesion between the two scaffold layers but also improve biological properties of scaffolds. It was demonstrated that functionalized bilayer scaffolds could promote cell adhesion, spreading and proliferation of mouse preosteoblastic MC3T3-E1 cells and rat bone-marrow-derived stromal cells (rBMSCs). Furthermore, immunofluorescence staining and calcium deposition studies revealed that functionalized bilayer scaffolds could enhance osteogenic differentiation of MC3T3-E1 cells and rBMSCs in comparison with simple electrospun PLGA scaffolds. The functionalized bilayer scaffolds are promising structures for bone tissue engineering.

Three-Dimensional-Printable Thermo/Photo-Cross-Linked Methacrylated Chitosan-Gelatin Hydrogel Composites for Tissue Engineering.

Biomimetic constructs imitating the functions, structures, and compositions of normal tissues are of great importance for tissue repair and regeneration. Three-dimensional (3D) printing is an innovative method to construct intricate biomimetic 3D tissue engineering scaffolds with spatiotemporal deposition of materials to control the intrinsic architectural organization and functional performance of the scaffold. However, due to the lack of bioinks with suitable printability, high structural integrity, and biological compatibility, producing constructs that mimic the anisotropic 3D extracellular environments remains a challenge. Here, we present a printable hydrogel ink based on methylacrylate-modified chitosan (ChMA) and gelatin (GelMA) embedding nanohydroxyapatite (nano-Hap). This polymer composite is first physically cross-linked by thermal gelation for postprinting structural stability, followed by covalent photo-cross-linking of ChMA and GelMA to form a long-term stable structure. The rheological behavior of the hydrogels and the mechanical strengths of the printed constructs are tuned by adjusting the content of GelMA, which in turn enhances the shape retention after printing and enables the precise deposition of multilayered 3D scaffolds. Moreover, the formulated biomaterial inks exhibit biological characteristics that effectively support the spreading and proliferation of stem cells seeded on the scaffolds after 7 days of in vitro culture. Adding Hap has minor influences on the mechanical rigidity and cytocompatibility of the hydrogels compared with the group free of Hap. Together, the printable biomaterial inks with shear thinning and good structural integrity, along with biological cues, are promising for tissue engineering application.

Development of Multi-layer Tubular Vascular Scaffold to Enhance Compliance by Exhibiting a Negative Poisson’s Ratio

Synthetic small diameter vascular grafts frequently fail owing to intimal hyperplasia, results from mismatched compliance between the vascular graft and native vessels. A vascular graft with a negative Poisson's ratio (NPR, materials expand transversely when pulled axially) was suggested to enhance compliance. We produced a three-layer tubular vascular scaffold with NRP properties. The luminal side consisted of nanosized electrospun fibers for endothelial cell (EC) growth. The middle layer was an NPR structure created using 3D printing, and the outer layer was a microsized electrospun fiber layer for vascular smooth muscle cell (VSMC) growth. The developed multi-layer tubular vascular scaffold contained NPR value. And the NPR vascular scaffold showed 1.7 time higher compliance than the PPR scaffold and 3.8 times higher than that of commercial polytetrafluoroethylene (PTFE) vascular graft. In addition, the ECs and VSMCs were well survived and proliferated on the scaffold during 10 days of culture. From the optimized co-culture condition of the VSMCs and ECs that VSMC phenotype changed was inhibited, we successfully generated a thin luminal layer, which consisted of ECs and the proper thickness of the VSMC layer under the ECs. This scaffold may have a potential to replace conventional artificial vascular graft by providing enhanced compliance and improved cell culture environment.

Preparation of multilayer electrospun nanofibrous scaffolds containing soluble eggshell membrane as potential dermal substitute.

Electrospinning of natural and synthetic polymers has shown to be a great candidate for the fabrication of tissue engineering scaffolds due to their similarity to the nanofibrous structure of natural extracellular matrix (ECM). Moreover, the addition of ECM-like proteins could enhance the biocompatibility of these scaffolds. In this study, soluble eggshell protein (SEP) was first extracted and synthesized from the raw eggshell membrane. The characteristics and biocompatibility of the extracted SEP were evaluated using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) analysis and 3-(4,5- dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) (MTT) assay. For scaffolds fabrication, a three-layer nanofibrous composite structure was produced using the electrospinning technique. The outer layers composed of polyvinyl alcohol, chitosan, and extracted SEP while the middle layer composed of polyethylene oxide, gelatin, and zinc oxide nanoparticles (ZnO-NPs). For each layer, the electrospinning parameters were adjusted to form bead-free fibers. To improve fibers' stability against body fluids, the produced fibers were crosslinked using glutaraldehyde vapor. Several techniques such as scanning electron microscopy (SEM), energy dispersive X-ray, ATR-FTIR, swelling, tensile test, in vitro biodegradation, and MTT assay were implemented to evaluate the physical, chemical, and biological characterization of the fabricated fibers. The results showed that crosslinked fibers have adequate stability in water, suitable mechanical properties, and promising water uptake capacity. The MTT results also revealed that SEP and ZnO-NPs could increase scaffolds biocompatibility. Moreover, SEM photographs of cultured fibroblasts cells on the scaffolds showed that cells were well attached on the scaffolds and preserve their natural spindle shapes. Altogether, our findings demonstrated that the produced three-layer composite scaffolds are potential candidates for skin tissue engineering.

3D multiphasic porous scaffolds of calcium phosphates doping with silicon and magnesium

In this work it is demonstrated the capacity of the sol–gel process to obtain materials with different phases, using the same chemical composition, and modifying only the conditions of hydrolysis and condensation. This method combined with the polymer sponge replica technique allowed to obtain a porous and three-dimensional scaffold, called core, consisting mainly of calcium phosphates. Additionally, due to the versatility of the sol–gel process, this core was coated with additional phases, constituted by silicon and magnesium, obtaining multilayer scaffolds. The scaffolds were physically and chemically characterized and the in vitro bioactivity was evaluated by immersion in simulated body fluid. The results of in vitro bioactivity showed that the core revealed a lamellar microstructure with some apatite precipitates after 14 days, while the multilayer scaffolds presented precipitates over the whole surface in only 3 days. The accelerated bioactivity and the general properties of the multiphase and multilayer scaffolds show the potential of this material in the area of tissue engineering.

Hierarchical multi-layered scaffolds based on electrofluidodynamic processes for tissue engineering

The aim of this study was to obtain hierarchical scaffolds combining 3D printing and two electrofluidodynamic methods. The multi-layered scaffold is composed by 3D printed struts, electrospun fibers obtained from poly(ϵ-caprolactone) and electrosprayed spheres produced from hydrophobically modified chitosan, namely chitosan grafted with linoleic acid (CHLA). Since CHLA has been used for the first time in the electrospraying (electro dynamic spraying, EDS) process, the formation of spheres needed an optimization process. The EDS process was strongly affected by the solvent mixture composition, concentration of acid used for CHLA dissolution and solution flow rate. By using the optimized electrospraying conditions, uniformly distributed spheres have been obtained, decorating struts and nanofibers. Preliminary biological tests with mouse preosteoblasts (MC3T3-E1) were performed to investigate the effect of the hierarchical scaffold on cell seeding efficacy. Results showed that the hierarchical structure enhances cell seeding efficacy, respect to the 3D printed struts alone, preventing that the cells passed through the struts during the seeding. Moreover, the addition of the electrosprayed nanoparticles does not affect the cell seeding efficiency. The versatility of the proposed structure, with the added value of CHLA nanoparticles decoration could be suitable for several applications in tissue engineering, mainly related to drug delivery systems.

Epidermal and fibroblast growth factors incorporated polyvinyl alcohol electrospun nanofibers as biological dressing scaffold

In this study, single, mix, multilayer Polyvinyl alcohol (PVA) electrospun nanofibers with epidermal growth factor (EGF) and fibroblast growth factor (FGF) were fabricated and characterized as a biological wound dressing scaffolds. The biological activities of the synthesized scaffolds have been verified by in vitro and in vivo studies. The chemical composition finding showed that the identified functional units within the produced nanofibers (O–H and N–H bonds) are attributed to both growth factors (GFs) in the PVA nanofiber membranes. Electrospun nanofibers' morphological features showed long protrusion and smooth morphology without beads and sprayed with an average range of 198–286 nm fiber diameter. The fiber diameters decrement and the improvement in wettability and surface roughness were recorded after GFs incorporated within the PVA Nanofibers, which indicated potential good adoption as biological dressing scaffolds due to the identified mechanical properties (Young’s modulus) in between 18 and 20 MPa. The MTT assay indicated that the growth factor release from the PVA nanofibers has stimulated cell proliferation and promoted cell viability. In the cell attachment study, the GFs incorporated PVA nanofibers stimulated cell proliferation and adhered better than the PVA control sample and presented no cytotoxic effect. The in vivo studies showed that compared to the control and single PVA-GFs nanofiber, the mix and multilayer scaffolds gave a much more wound reduction at day 7 with better wound repair at day 14–21, which indicated to enhancing tissue regeneration, thus, could be a projected as a suitable burn wound dressing scaffold.

A competitive nature-derived multilayered scaffold based on chitosan and alginate, for full-thickness wound healing

The aim of this study was to evaluate a bioactive multilayer wound dressing, based on chitosan and alginate. To enhance healing potential, Dracaena Cinnabari and Aloe Vera were loaded as separate layers into the scaffold. The bare and bioactive multilayered scaffolds were fabricated by an iterative layering freeze-drying technique. Following of topographical, chemical, and physical assessment, the performance of the scaffolds was evaluated in vitro and in vivo. The results revealed adequate attachment, and proliferation of human foreskin fibroblasts, indicating excellent biocompatibility of the bioactive scaffold. In vivo, the performance of the multi-layered scaffold loaded with the bioactive materials was comparable with Comfeel plus®. The wounds treated with the bioactive scaffold exhibited superior hypergranulation, fibroblast maturation, epithelization, and collagen deposition, with minimal inflammation, and crust formation. It is concluded that the synergism of extracellular matrix-mimicking multi-layered scaffolding with Aloe Vera and Dracaena Cinnabari could be considered as a supportive wound dressing.

Gradient 3D Printed PLA Scaffolds on Biomedical Titanium: Mechanical Evaluation and Biocompatibility.

The goal of the present investigation was to find a solution to crucial engineering aspects related to the elaboration of multi-layered tissue-biomimicking composites. 3D printing technology was used to manufacture single-layered and gradient multi-layered 3D porous scaffolds made of poly-lactic acid (PLA). The scaffolds manufacturing process was optimized after adjusting key printing parameters. The scaffolds with 60 μm side length (square-shaped pores) showed increased stiffness values comparing to the other specimens. A silicone adhesive has been further used to join biomedical titanium plates, and the PLA scaffolds; in addition, titania nanotubes (TNTs were produced on the titanium for improved adhesion. The titanium-PLA scaffold single lap joints were evaluated in micro-tensile testing. The electrochemical processing of the titanium surface resulted in a 248% increase of the ultimate strength in the overlap area for dry specimens and 40% increase for specimens immersed in simulated body fluid. Finally, the biocompatibility of the produced scaffolds was evaluated with primary cell populations obtained after isolation from bone residual tissue. The manufactured scaffolds present promising features for applications in orthopedic implantology and are worth further.

Novel Self-Directing Single-Polymer Jet Developing Layered-Like 3D Buckled Microfibrous Scaffolds for Tissue Engineering Applications.

Electrospinning is a promising technique for the fabrication of bioscaffolds in tissue engineering applications. Pertaining issues of multiple polymer jets and bending instabilities result in random paths which lend poor controllability over scaffolds morphology for affecting the porosity and mechanical stability. The present study alleviates these challenges by demonstrating a novel self-directing single jet taking a specifically patterned path to deposit fibers into circular and uniform scaffolds without tuning any externally controlled parameters. High-speed camera observation revealed that the charge retention and dissipation on the collected fibers caused rapid autojet switching between the two jetting modes, namely, a microcantilever-like armed jet motion and a whipping motion, which sequentially expand the area and thickness of the scaffolds, respectively, in a layered-like fashion. The physical properties showed that the self-switching dual-jet modes generated multilayered microfibrous scaffolds (MFSs) with dual morphologies and varied fiber packing density, thereby establishing the gradient porosity and mechanical strength (through buckled fibers) in the scaffolds. In vitro studies showed that as-spun scaffolds are cell-permeable hierarchical 3D microporous structures enabling lateral cell seeding into multiple layers. The cell proliferation on days 6 and 9 increased 21% and 38% correspondingly on MFSs than on nanofibrous scaffolds (NFSs) done by conventional multijets electrospinning. Remarkably, this novel and single-step process is highly reproducible and tunable for developing fibrous scaffolds for tissue engineering applications.

Chitosan/polycaprolactone multilayer hydrogel: A sustained Kartogenin delivery model for cartilage regeneration

Cartilage regeneration using biomaterial-guided delivery systems presents improved therapeutic efficacy of the biomolecules while minimizing side effects. Here, our hypothesis was to design a multilayer scaffold of chitosan (CS) hydrogel and polycaprolactone (PCL) mat to enhance the mechanical properties, integrity and stability of CS, especially for subsequent in vivo transplantation. After conjugation of the Kartogenin (KGN) into this structure, its gradual release can promote chondrogenesis of mesenchymal stem cells (MSCs). Initially, a thin electrospun PCL layer was sandwiched between two CS hydrogels. Subsequently, KGN was superficially immobilized onto the CS matrix. The successful conjugation was confirmed by scanning electron microscopy (SEM) and infrared spectroscopy. These novel KGN-conjugated scaffolds possessed lower swelling and higher compressive modulus and showed gradual release of KGN in longer retention times. Immunofluorescent and histological staining represented more cells located in lacunae as well as more Coll2 and Sox9 positive cells on KGN-conjugated scaffolds. Gene expression analysis also revealed that SOX9, COLL2 and ACAN expression levels were higher in the presence of KGN, while COLLX expression was down-regulated, indicating a hypertrophy phenomenon with synergistic effect of TGF-β. This multilayer structure not only facilitates the effective treatment, but also provides a proper mechanical structure for cartilage engineering.

Multilayer Mg-pyrophosphate glass ceramic with discontinuous bioactivity. Physicochemical characterization

Novel pyrophosphate glass ceramic scaffolds (3D-Ca-P2) were fabricated and coated with several layers of CaO-P2O5-SiO2-MgO [3D-[Ca/P/Si]-XMg (X = 1,3 and 10 mol%)] as potential bioactive scaffolds for bone regeneration. The core scaffolds present a homogeneous distribution of polygonal grains consisting of 97% Ca2P2O7 with CaO-P2O5 glass as the matrix phase. Incorporating MgO into coatings led to extremely porous layers, assembled by nanosized equiaxed particles, which favoured the formation of different phases of tricalcium phosphate instead of pyrophosphate. Possible applications were determined based on Standard ISO/FDIS 23317:2014 for ceramics and powder samples. The 3D-Ca-P2 scaffolds’ non-bioactivity was modulated by coatings of different bioactivity behaviours depending on MgO contents. 3D-[Ca/P/Si]-10Mg was the best scaffold as precipitated hydroxyapatite (HA), was more amorphous than in the other scaffolds because Mg was incorporated into the HA network, which could favour subsequent cell adhesion. The multilayer scaffold provides advantages over others developed until now because in a unique structure all the characteristics required for an implant are combined. On a physical level, mechanical resistance, and porosity, and on a chemical level, a combination of calcium phosphate phases, similar to the mineral phase of bone, doped with Mg ions, which are capable of being transformed into HA in different periods of time.

A novel polymeric fibrous microstructured biodegradable small-caliber tubular scaffold for cardiovascular tissue engineering

Increasing morbidity of cardiovascular diseases in modern society has made it crucial to develop artificial small-caliber cardiovascular grafts for surgical intervention of diseased natural arteries, as alternatives to the gold standard autologous implants. Synthetic small-caliber grafts are still not in use due to increased risk of restenosis, lack of lumen re-endothelialization and mechanical mismatch, leading sometimes either to graft failure or to unsuccessful remodeling and pathology of the distal parts of the anastomosed healthy vascular tissues. In this work, we aimed to synthesize small-caliber polymeric (polycaprolactone) tissue-engineered vascular scaffolds that mimic the structure and biomechanics of natural vessels. Electrospinning was implemented to prepare microstructured polymeric membranes with controlled axis-parallel fiber alignment. Consequently, we designed small-caliber multilayer anisotropic biodegradable nanofibrous tubular scaffolds, giving attention to their radial compliance. Polycaprolactone scaffold morphology and mechanical properties were assessed, quantified, and compared with those of native vessels and commercial synthetic grafts. Results showed a highly hydrophobic scaffold material with a three-layered tubular morphology, 4-mm internal diameter/0.25 ± 0.09-mm thickness, consisting of predominantly axially aligned thin (1.156 ± 0.447 μm), homogeneous and continuous microfibers, with adequate (17.702 ± 5.369 μm) pore size, potentially able to promote cell infiltration in vivo. In vitro accelerated degradation showed a 5% mass loss within 17–25 weeks. Mechanical anisotropy was attained as a result, almost one order of magnitude difference of the elastic modulus (18 ± 3 MPa axially/1 ± 0.3 MPa circumferentially), like that of natural arterial walls. Furthermore, a desirable radial compliance (5.04 ± 0.82%, within the physiological pressure range) as well as cyclic stability of the tubular scaffold was achieved. Finally, cytotoxicity evaluation of the polymeric scaffolds revealed that the materials were nontoxic and did not release substances harmful to living cells (over 80% cell viability achieved).

Electric-field-driven jet deposition micro-nano 3D printing based on a single-plate electrode

<p indent="0mm">Micro/nano-scale 3D printing has become one of the most popular research topics of additive manufacturing due to its wide range of applications in biological tissue engineering, flexible electronics, new energy, new materials, microelectromechanical systems, and many other fields. Recent work on micro/nano-scale 3D printing has presented a series of advanced techniques, such as microstereolithography, two-photon stereolithography, micro-laser sintering, electrochemical fabrication, and electrohydrodynamic (EHD) jet printing, to directly fabricate 3D micro/nano-scale structures. However, these existing technologies are still faced with challenges in realizing multi-material, macro/micro multi-scale 3D printing. Here, we propose a new electric-field-driven jet deposition micro/nano-scale 3D printing based on a single-plate electrode. Differing from the traditional EHD printing with two counter electrodes and our previously proposed electric-field-driven jet deposition 3D printing with a single nozzle electrode, this 3D printing is achieved using technology based on a self-induced electrostatic field. In this method, the nozzle is no longer used as the electrode, and only a single-plate electrode is needed to connect to the positive electrode of high voltage power supply while the negative electrode is directly grounded, which not only overcomes the mandatory requirement of nozzle conductivity in traditional EHD jet printing, but also solves the discharge and breakdown problem of printing conductive materials on the conductive substrate. The micro/nano-scale additive manufacturing by this proposed method can be achieved by combining the necking effect of Taylor cone formed by the self-induced electrostatic field between the printing material on the nozzle tip and the top surface of the substrate, and multi-layer precise stacking by the polarization charges attraction between the printing material and the already printed materials on the substrate. In addition, considering the high-resolution and high-efficiency printing of various materials with different viscosities, we propose two working modes, including the pulsed cone-jet mode and the continuous cone-jet mode. To prove the advantages and features of the proposed method, we carried out a series of research work systematically. Firstly, the printing mechanism is revealed through theoretical analysis and numerical simulation. The higher the conductivity of the single-plate electrode is and the lower the conductivity of the nozzle is, the greater the electric field intensity is. Then, the feasibility of printing with the nozzle (conductive steel nozzle and non-conductive glass nozzle), substrate (conductive copper plate, semiconductor silicon wafer, and insulating glass plate), and printing material (conductive silver paste and non-conductive polymer) has been verified by systematic experiments. Finally, three typical cases, micro “wall” structure of polylactic acid (PLA) with a line width of <sc>1.139 μm</sc> and a high aspect ratio of 46.8:1, high-performance (transmittance of 90.17% and sheet resistance of 4.26 Ω/sq) transparent electrode made of high viscosity silver paste, and multi-layer 3D scaffold with a line width of <sc>20 µm</sc> and a total height of <sc>200 µm,</sc> have been printed successfully. The new method has been proved to have unique technical advantages in high-resolution printing, multi-material, and macro/micro multi-scale printing. Therefore, it provides a new solution with low cost and high universality for micro/nano-scale additive manufacturing and macro/micro cross scale 3D printing, especially in the field of biological tissue engineering and printing electronics.

A Graded, Porous Composite of Natural Biopolymers and Octacalcium Phosphate Guides Osteochondral Differentiation of Stem Cells.

Lesions involving the osteochondral unit are difficult to treat. Biomimetic scaffolds are previously shown as promising alternative. Such devices often lack multiple functional layers that mimic bone, cartilage, and the interface. In this study, multilayered scaffolds are developed based on the use of natural extracellular matrix (ECM)-like biopolymers. Particular attention is paid to obtain a complex matrix that mimics the native osteochondral transition. Porous, sponge-like chitosan-collagen-octacalcium phosphate (OCP) scaffolds are obtained. Collagen content increases while the amount of OCP particles decreases toward the cartilage layer. The scaffolds are bioactive as a mineral layer is deposited containing hydroxyapatite at the bony side. The scaffolds stimulate proliferation of human adipose-derived mesenchymal stem cells, but the degree of proliferation depends on the cell seeding density. The scaffolds give rise to a zone-specific gene expression. RUNX2, COL1A1, BGLAP, and SPP1 are upregulated in the bony layer of the scaffold. SOX9 is upregulated concomitant with COL2A1 expression in the cartilage zone. Mineralization in presence of the cells is prominent in the bone area with Ca and P steadily increasing over time. These results are encouraging for the fabrication of biomimetic scaffolds using ECM-like materials and featuring gradients that mimic native tissues and their interface.

Fabrication of multilayered electrospun poly(lactic-co-glycolic acid)/polyvinyl pyrrolidone + poly(ethylene oxide) scaffolds and biocompatibility evaluation.

Poly(lactic-co-glycolic acid)/polyvinyl pyrrolidone + poly(ethylene oxide) [PLGA/(PVP + PEO)] scaffolds with different polymer concentrations were fabricated using multilayered electrospinning, and their physicochemical properties and biocompatibility were examined to screen for scaffolds with excellent performance in tissue engineering (TE). PLGA solution (15% w/v) was used as the bottom solution, and a mixed solution of 12% w/v PVP + PEO was applied as the surface layer solution. The mass ratios of PVP vs. PEO in each 10ml surface layer mixed solution were 1.08 g: 0.12 g; 0.96 g: 0.24 g; and 0.84 g: 0.36 g. Compared to the conventional electrospinning method used to fabricate the pure PVP + PEO (0.96 g: 0.24 g, Group A) scaffold and pure PLGA (Group E) scaffold, the multilayer electrospinning technique of alternating sprays of the bottom layer solution and the surface layer solution was adopted to fabricate multilayer nanofiber scaffolds, including PLGA/(PVP + PEO) (1.08 g: 0.12 g, Group B), PLGA/(PVP + PEO) (0.96 g: 0.24 g, Group C), and PLGA/(PVP + PEO) (0.84 g: 0.36 g, Group D). The morphology and characteristics of the five scaffolds were analyzed, and the biocompatibilities of the cell-scaffold composites were assessed through methods including Cell Counting Kit-8 (CCK8) analysis, 4',6-diamidino-2-phenylindole (DAPI) staining, and scanning electron microscopy. Therefore, with a PVP-to-PEO mass ratio of 0.96 g: 0.24 g, an optimal multilayer nanofiber scaffold was fabricated by the multilayer electrospinning technique. The excellent biocompatibility and mechanical properties of the scaffold were confirmed by in vitro experiments, which demonstrated the scaffold's promising application potential in the field of TE.

3D printing of an integrated triphasic MBG-alginate scaffold with enhanced interface bonding for hard tissue applications.

Osteochondral defects affect both of cartilage and subchondral areas, thus it poses a significant challenge to simultaneously regenerate two parts in orthopedics. Tissue engineering strategy is currently regarded as the most promising way to repair osteochondral defects. This study focuses on developing a multilayered scaffold with enhanced interface bonding through 3D printing. One-shot printing process enables control over material composition, pore structure, and size in each region of the scaffold, while realizes seamlessly integrated construct as well. The scaffold was designed to be triphasic: a porous bone layer composed of alginate sodium (SA) and mesoporous bioactive glasses (MBG), an intermediate dense layer also composed of SA and MBG and a cartilaginous layer composed of SA. The mechanical strength including the interface adhesion strength between layers were characterized. The results indicated that SA crosslinking after 3D printing anchored different materials together and integrated all regions. Additional scaffold soaking in simulated body fluid (SBF) and cell culture medium induced apatite deposition and had weakened the compressive and tensile strengths, while no layer dislocation or delamination occurred.