Biofabrication最新文献

Inkjet printing techniques are often used for bioprinting purposes because of their excellent printing characteristics, such as high cell viability and low apoptotic rate, contactlessmodus operandi, commercial availability, and low cost. However, they face some disadvantages, such as the use of bioinks of low viscosity, cell damage due to shear stress caused by drop ejection and jetting velocity, as well as a narrow range of available bioinks that still challenge the inkjet printing technology. New technological solutions are required to overcome these obstacles. Pneumatic conveying printing, a new type of inkjet-based printing technique, was applied for the bioprinting of both acellular and cellular fibrin-hydrogel droplets. Drops of a bioink containing 6 × 10⁶HEK293H cells ml^-1were supplied from a sterile nozzle connected to a syringe pump and deposited on a gas stream on a fibrinogen-coated glass slide, here referred to as biopaper. Fibrinogen film is the substrate of the polymerization reaction with thrombin and Ca²⁺present in the bioink. The pneumatic conveying printing technique operates on a mechanism by which drop ejection and deposition in a stream of gas occurs. The percentage of unprinted and printed dead HEK293H cells was 5 ± 2% and 7 ± 4%, respectively. Thus, compared to normal handling, pneumatic conveying printing causes only little damage to the cells. The velocity of the drop approaching the biopaper surface is below 0.2 m s^-1and does not cause any damage to the cells. The cell viability of printed cells was 93%, being an excellent value for inkjet printing technology. The HEK293H cells exhibited approximately a 24 h lag time of proliferation that was preceded by intense migration and aggregation. Control experiments proved that the cell migration and lag time were associated with the chemical nature of the fibrin hydrogel and not with cell stress.

Volumetric bioprinting has revolutionized the field of biofabrication by enabling the creation of cubic centimeter-scale living constructs at faster printing times (in the order of seconds). However, a key challenge remains: developing a wider variety of available osteogenic bioinks that allow osteogenic maturation of the encapsulated cells within the construct. Herein, the bioink exploiting a step-growth mechanism (norbornene-norbornene functionalized gelatin in combination with thiolated gelatin-GelNBNBSH) outperformed the bioink exploiting a chain-growth mechanism (gelatin methacryloyl-GelMA), as the necessary photo-initiator concentration was three times lower combined with a more than 50% reduction in required light exposure dose resulting in an improved positive and negative resolution. To mimic the substrate elasticity of the osteoid, two concentrations of the photo-initiator Li-TPO-L (1 and 10 mg ml^-1) were compared for post-curing whereby the lowest concentration was selected since it resulted in attaining the osteogenic substrate elasticity combined with excellent biocompatibility with HT1080 cells (>95%). Further physico-chemical testing revealed that the volumetric printing (VP) process affected the degradation time of the constructs with volumetric constructs degrading slower than the control sheets which could be due to the introduced fibrillar structure inherent to the VP process. Moreover, GelNBNBSH volumetric constructs significantly outperformed the GelMA volumetric constructs in terms of a 2-fold increase in photo-crosslinkable moiety conversion and a 3-fold increase in bulk stiffness of the construct. Finally, a 21-day osteogenic cell study was performed with highly viable dental pulp-derived stem cells (>95%) encapsulated within the volumetric printed constructs. Osteogenesis was greatly favored for the GelNBNBSH constructs through enhanced early (alkaline phosphatase activity) and late maturation (calcium production) osteogenic markers. After 21 d, a secretome analysis revealed a more mature osteogenic phenotype within GelNBNBSH constructs as compared to their chain-growth counterpart in terms of osteogenic, immunological and angiogenic signaling.

3D bioprinting of plant cells has emerged as a promising technology for plant cell immobilization and related applications. Despite the numerous progress in mammal cell printing, the bioprinting of plant cells is still in its infancy and needs further investigation. Here, we present a systematic study on optimizing the 3D bioprinting of plant cells, using carrots as an example, towards enhanced resolution and cell viability. We mainly investigated the effects of cell cluster forms and nozzle size on the rheological, extrusion, and printability properties of plant cell bioinks, as well as on the resultant cell viability and growth. We found that when the printing nozzle is larger than 85% of the cell clusters embedded in the bioink, smooth extrusion, and good printability can be achieved together with considerable cell viability and long-term growth. Specifically, we optimized a bioink composited with suspension-cultured carrot cells, which exhibited better transparency, smoother extrusion, and higher cell viability over a one-month culture compared to those with the regular callus or fragmented callus. This work provides a practical guideline for optimizing plant cell bioprinting from the bioink development to the printing outcome assessment. It highlights the importance of selecting a matched nozzle and cell cluster and might provide insights for a better understating and exploitation of plant cell bioprinting.

The design and development of advanced surgical sutures with appropriate structure and abundant bio-functions are urgently required for the chronic wound closure and treatment. In this study, an integrated technique routine combining modified electrospinning with hot stretching process was proposed and implemented to fabricate poly(L-lactic acid) (PLLA) nanofiber sutures, and the Salvia miltiorrhiza Bunge-Radix Puerariae herbal compound (SRHC) was encapsulated into PLLA nanofibers during the electrospinning process to enrich the biofunction of as-generated sutures. All the PLLA sutures loading without or with SRHC were found to exhibit bead-free and highly-aligned nanofiber structure. The addition of SRHC was found to have no significant influences on the fiber morphology, diameter, and the crystallinity of as-prepared PLLA sutures. Importantly, all the SRHC-contained PLLA nanofiber sutures possessed excellent tensile and knot strength, which were of significant importance for the surgical suture applications. Besides, the antioxidant and anti-inflammatory properties of these sutures obviously enhanced with the increasing of SRHC concentration. Furthermore, the in vitro cell tests illustrated that the high fiber orientation of the sutures was able to efficiently induce the human dermal fibroblasts (HDFs) to migrate in a rapid manner, and the sutures loaded with high content of SRHC could significantly promote the attachment and proliferation of HDFs in comparison. The in vivo diabetic mouse model experiments revealed that all the as-developed PLLA sutures could effectively close the wound, but the PLLA sutures containing high content of SRHC could dramatically promote the wound healing with high quality by shortening the healing time, improving the collagen deposition, neovascularization, and the regeneration of hair follicles, especially compared with commercial polyester (PET) suture. This study offers a simple and easily-handling strategy to develop robust, biodegradable, bioactive, and nanostructured PLLA sutures, which shows huge potential for the treatment of hard-to-heal diabetic wounds. .

Producing oral soft tissues using tissue engineering could compensate for the disadvantages of autologous grafts (limited availability and increased patient morbidity) and currently available substitutes (shrinkage). However, there is a lack of in vitro-engineered oral tissues due to the difficulty of obtaining stable pre-vessels that connect to the host and enable graft success. The main objective was to assess the connection of pre-vascularised 3D-bioprinted gingival substitutes to the host vasculature when subcutaneously implanted in immunodeficient mice. This study produced vascularised connective tissue substitutes using extrusion-based 3D-bioprinting of primary human gingival fibroblasts (hGF) and fluorescent human endothelial cells (RFP-HUVEC) cocultures. Pre-vascularised (hGF+RFP-HUVEC -CC grids) and control (hGF only -HG grids) grids were bioprinted and pre-cultivated for 14 days to enable pre-vessels formation. In vitro vessel formation follow-up was performed. Eight-week-old female NOG mice were used for in vivo experiments. One grid per mouse was subcutaneously implanted in 20 mice (10HG/10CC). The fluorescent activity of RFP-HUVEC was monitored. Samples were retrieved at 7, 14 and 21 days. Histological, immunohistochemical, and immunofluorescent staining was performed. CC-grids formed efficient and stable pre-vessel networks within 14 days of static pre-culture. HG-grids did not contain any vessel, while CC-grids successfully connected to the host vasculature by presenting erythrocytes within the vessel lumen inside the grids starting day 7. From days 7 to 21, vessel density was stable. Human pre-vessels were present at 7 days and were progressively replaced by murine endothelial cells. This study showed that primary hGF-HUVEC co-cultures can be successfully 3D-bioprinted within biomimetic hydrogels having a close composition to the gingival connective tissue, and HUVEC organise themselves into pre-vessel networks that connect to the murine vasculature when implanted in vivo. This approach represents a promising strategy to enhance current and future oral soft tissue substitutes for prospective clinical applications.

Advances in biofabrication have enabled the generation of freeform perfusable networks mimicking vasculature. However, key challenges remain in the effective endothelialization of these complex, vascular-like networks, including cell uniformity, seeding efficiency, and the ability to pattern multiple cell types. To overcome these challenges, we present an integrated fabrication and endothelialization strategy to directly generate branched, endothelial cell-lined networks using a diffusion-based, embedded 3D bioprinting process. In this strategy, a gelatin microparticle sacrificial ink delivering both cells and crosslinkers is extruded into a crosslinkable gel precursor support bath. A self-supporting, perfusable structure is formed by diffusion-induced crosslinking, after which the sacrificial ink is melted to allow cell release and adhesion to the printed lumen. This approach produces a uniform cell lining throughout networks with complex branching geometries, which are challenging to uniformly and efficiently endothelialize using conventional perfusion-based approaches. Furthermore, the biofabrication process enables high cell viability (>90%) and the formation of a confluent endothelial layer providing vascular-mimetic barrier function and shear stress response. Leveraging this strategy, we demonstrate for the first time the patterning of multiple endothelial cell types, including arterial and venous cells, within a single arterial-venous-like network. Altogether, this strategy enables the fabrication of multi-cellular engineered vasculature with enhanced geometric complexity and phenotypic heterogeneity.

Corneal blindness, a leading cause of visual impairment globally, has created a pressing need for alternatives to corneal transplantation due to the severe shortage of donor tissues. In this study, we present a novel interpenetrating network hydrogel composed of gelatin methacryloyl (GelMA) and oxidized carboxymethyl cellulose (OxiCMC) for bioprinting a biomimetic corneal stroma equivalent. We tested different combinations of GelMA and OxiCMC to optimize printability and subsequently evaluated these combinations using rheological studies for gelation and other physical, chemical, and biological properties. Using digital light processing (DLP) bioprinting, with tartrazine as a photoabsorber, we successfully biofabricated three-dimensional constructs with improved shape fidelity, high resolution, and excellent reproducibility. The bioprinted constructs mimic the native corneal stroma's curvature, with central and peripheral thicknesses of 478.9 ± 56.5 µm and 864.0 ± 79.3 µm, respectively. The dual crosslinking strategy, which combines Schiff base reaction and photocrosslinking, showed an improved compressive modulus (106.3 ± 7.7 kPa) that closely matched that of native tissues (115.3 ± 13.6 kPa), without relying on synthetic polymers, toxic crosslinkers, or nanoparticles. Importantly, the optical transparency of tartrazine-containing corneal constructs was comparable to the native cornea following phosphate-buffered saline washing. Morphological analyses using scanning electron microscopy confirmed the improved porosity, interconnected network, and structural integrity of the GelMA-OxiCMC hydrogel, facilitating better nutrient diffusion and cell viability. In vitro biological assays demonstrated high cell viability (>93%) and desirable proliferation of human corneal keratocytes within the biofabricated constructs. Our findings indicate that the GelMA-OxiCMC hydrogel system for DLP bioprinting presents a promising alternative for corneal tissue engineering, offering a potential solution to the donor cornea shortage and advancing regenerative medicine for corneal repair. .

The process of micromachining has garnered attention for its ability to create three-dimensional tiny features, particularly in ultra-hard and exotic materials. The present work investigates the effect of different parameters of the µ-ED milling, such as pulse on time (Ton), pulse off time (Toff), voltage (V), and tool rotation (TR) on the dimensional deviation (DD), material removal rate (MRR), surface roughness (Ra), and machined surface characteristics (analysed by EDS and FESEM). The sesame oil as dielectric and tungsten-copper as tool electrodes were used to maintain the accuracy and improve the machinability of bio-grade Nitinol SMA. Response surface methodology (RSM) and genetic algorithms (GA) were used to optimize the various input parameters of the µ-ED milling process. Artificial neural network (ANN) was combined with GA to find the best parametric combination for microchannel fabrication. The cytotoxicity test was also performed on the machined surface to analyse the biocompatibility of the machined surface. It was found that the cell viability of Nitinol SMA was improved by 85.11% after machining at the optimum condition. The highest MRR was found to be 0.076 gm/min, and the lowest DD and Ra were found to be 16.47 µm and Ra 0.387µm, respectively.

Despite significant advances in bioprinting technology, current hardware platforms lack the capability for process monitoring and quality control. This limitation hampers the translation of the technology into industrial GMP-compliant manufacturing settings. As a key step towards a solution, we developed a novel bioprinting platform integrating a high-resolution camera for in-situ monitoring of extrusion outcomes during embedded bioprinting. Leveraging classical computer vision and image analysis techniques, we then created a custom software module for assessing print quality. This module enables quantitative comparison of printer outputs to input points of the CAD model's 2D projections, measuring area and positional accuracy. To showcase the platform's capabilities, we then investigated compatibility with various bioinks, dyes, and support bath materials for both 2D and 3D print path trajectories. In addition, we performed a detailed study on how the rheological properties of granular support hydrogels impact print quality during embedded bioprinting, illustrating a practical application of the platform. Our results demonstrated that lower viscosity, faster thixotropy recovery, and smaller particle sizes significantly enhance print fidelity. This novel bioprinting platform, equipped with integrated process monitoring, holds great potential for establishing auditable and more reproducible biofabrication processes for industrial applications.

Three-dimensional (3D) bioprinting, an additive manufacturing technology, fabricates biomimetic tissues that possess natural structure and function. It involves precise deposition of bioinks, including cells, and bioactive factors, on basis of computer-aided 3D models. Articular cartilage injurie, a common orthopedic issue. Current repair methods, for instance microfracture procedure (MF), Autologous chondrocyte implantation (ACI), and Osteochondral Autologous Transfer Surgery (OATS) have been applied in clinical practice. However, each procedure has inherent limitation. For instance, microfracture surgery associates with increased subchondral cyst formation and brittle subchondral bone. ACI procedure involves two surgeries, and associate with potential risks infection and delamination of the regenerated cartilage. In addition, chondrocyte implantation's efficacy depends on the patient's weight, joint pathology, gender-related histological changes of cartilage, and hormonal influences that affect treatment and prognosis. So far, it is a still a grand challenge for achieving a clinical satisfactory in repairing and regeneration of cartilage defects using conditional strategies. 3D biofabrication provide a potential to fabricate biomimetic articular cartilage construct that has shown promise in specific cartilage repair and regeneration of patients. This review reported the techniques of 3D bioprinting applied for cartilage repair, and analyzed their respective merits and demerits, and limitations in clinical application. A summary of commonly used bioinks has been provided, along with an outlook on the challenges and prospects faced by 3D bioprinting in the application of cartilage tissue repair. It provided an overall review of current development and promising application of 3D biofabrication technology in articular cartilage repair.