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.

Craniofacial bone defect healing in periodontitis patients with diabetes background has long been difficult due to increased blood glucose levels which cause overproduction of reactive oxygen species (ROS) and a low pH environment. These conditions negatively affect the function of macrophages, worsen inflammation and oxidative stress, and ultimately, hinder osteoblasts' bone repair potential. In this study, we for the first time found that annexin A1 (ANXA1) expression in macrophages was reduced in a diabetic periodontitis (DP) environment, with the activation of the NLRP3/Caspase-1/GSDMD signaling pathway, and, eventually, increased macrophage pyroptosis. Next, we have developed a new GPPG intelligent hydrogel system which was ROS and pH responsive, and loaded with Ac2-26, an ANXA1 bioactive peptide, and osteogenic peptide OGP as well. We found that Ac2-26/OGP/GPPG can effectively reduce ROS, mitigates macrophage pyroptosis via the ANXA1/NLRP3/Caspase-1/GSDMD pathway and enhanced osteogenic differentiation. The effect of Ac2-26/OGP/GPPG in regulation of pyroptosis and bone defect repair was also further validated by animal experiments on periodontitis-induced tooth loss model in diabetic rats. To conclude, our study unveils the effect of ANXA1 on macrophage pyroptosis in periodontitis patients with diabetes, based on which we introduced a promising innovative hydrogel system for improvement of bone defects repair in DP patients via targeting macrophage pyroptosis and enhancing osteogenic potential.

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. .

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.

The objective of this review is to deepen understanding and emphasize scientific and technological progress in the transformation of crop by-products into bio-based dental materials. Amid heightened environmental sustainability consciousness, various sectors including dentistry have achieved novel advancements by utilizing bio-based materials from crop by-products for dental restorations. This paper provides a thorough review of the extraction, processing, and application of natural polymers, biopolymers, and bio-based mixtures at both the macroscopic and nanoscopic scales, with a focus on their contextualization within dental practices. The performance and efficacy of bio-resins, bio-sourced monomers, and biopolymers derived from these resources were scrutinized and compared with traditional petroleum-based counterparts. This study addresses the recycling and industrial valorization of bio-based dental materials, emphasizing their potential to foster a circular economy in dentistry.

Myocardial infarction (MI) remains a leading cause of mortality worldwide, posing a significant challenge to healthcare systems. The limited regenerative capacity of cardiac tissue following MI results in chronic cardiac dysfunction, highlighting the urgent need for innovative therapeutic strategies. In this study, we explored the application of a multidimensional nanofibrous hydrogel for myocardial regeneration. We developed a composite hydrogel system by integrating fibrin, polycaprolactone (PCL), and alginate. In this system, fibrin supported cell proliferation and significantly enhanced angiogenesis when combined with human umbilical vein endothelial cells (HUVECs). PCL contributed to the alignment of encapsulated cells, improving their organization within the scaffold. Adipose-derived stem cells (ADSCs) were encapsulated within the hydrogel for their versatile regenerative potential, while C2C12 cells were incorporated for their ability to form muscle tissue. Additionally, the inclusion of alginate not only enhanced the mechanical properties of the hydrogel to better match the biomechanical demands of cardiac tissue but also played a critical role in reducing the immune response, thereby improving the system's biocompatibility. This study presents an advanced platform for myocardial regeneration using a nanofibrous hydrogel system designed to meet the dual requirements of mechanical robustness and cellular compatibility essential for cardiac tissue engineering. The triculture system, consisting of ADSCs, C2C12 cells, and HUVECs, harnesses the regenerative capabilities of each cell type, promoting both angiogenesis and tissue regeneration. This comprehensive approach addresses the immediate needs for cellular survival and integration while effectively overcoming long-term mechanical and immunological challenges.