Biofabrication最新文献

3D-bioprinting is a promising technique to mimic the complex anatomy of natural tissues, as it comprises a precise and gentle way of placing bioinks containing cells and hydrogel. Although hydrogels expose an ideal growth environment due to their extracellular matrix (ECM)-like properties, high water amount and tissue like microstructure, they lack mechanical strength and possess a diffusion limit of a couple of hundred micrometers. Integration of electrospun fibers could hereby benefit in multiple ways, for instance by controlling mechanical characteristics, cell orientation, direction of diffusion and anisotropic swelling behavior. The aim of this study was to create an advanced ECM-biomimicking scaffold material for tissue engineering, which offers enhanced diffusion properties. PCL bulk membranes were successfully electrospun and fragmented using a cryo cutting technique. Subsequently, these short single fibers (<400µm in length and ∼5-10µm in diameter) were embedded in an agarose-based hydrogel after hydrophilization of the short single fibers by O₂plasma treatment. Fiber-filled bioinks exhibit significantly improved biomolecule diffusion (>500µm), swelling properties (20%-60% of control), and higher mechanical strength, while its viscosity (5-30 mPas*s) and gelation kinetics (28 °C) remained almost unaffected. The diffusion tests indicate a high level of size selectivity, which can be utilized for targeted biomolecule transport in the future. Finally, applying 3D-bioprinting technology (drop-on-demand vs. microextrusion) a print setting dependent post-dispensing orientation of the fibers could be induced, which ultimately paves way for the fabrication of metamaterials with anisotropic material properties. As expected, the fiber-filled bioink was found to be non-cytotoxic in cell culture trials using HUVECs and HepG2 (>80% viability). In summary, microfiber integration holds great promise for 3D-bioprinting of tissue percursors with advanced metamaterial properties and thus offers high applicability in various fields of research, such asin-vitrotissue models, tissue engineered implants or cultivated meat.

The fibroblast-myofibroblast transition marked by extracellular matrix (ECM) secretion and contraction of actomyosin-based stress fibers, plays central roles in the wound healing process. This work aims to utilize the cell membrane-based nanoplatform to improve the outcomes of dysregulated wound healing. The cell membranes of myofibroblasts isolated from mouse skin are used as the camouflage for gold nanoparticles loaded with IL-4 cytokine. The membrane-modified nanoparticles show effective in situ clearance of bacterial infection, and act as the activator in IL-4Rα signaling pathway to induce pro-inflammatory M1 macrophages into the anti-inflammatory M2 phenotype. Thus, the poor bacteria-clearance and non-stop inflammation in refractory wounds are improved and accelerated. Furthermore, the nanoplatform releases myofibroblast membranes to propel primitive fibroblasts toward the fibroblast-myofibroblast transition in an epigenetic manner. Matrix-production, vascularization, and epithelial regeneration are then initiated, leading to the satisfactory wound closure. Our study devises a new strategy for activating fibroblasts into myofibroblasts under prolonged and continuous exposure to the fibrotic environment, and develops a promising biomimetic nanoplatform for effective treatment of dysregulated chronic wound healing.

Reduced therapy response in breast cancer has been correlated with heterogeneity in biomarker composition, expression level, and spatial distribution of cancer cells within a patient tumor. Thus, there is a need for models to replicate cell-cell, cell-stromal, and cell-microenvironment interactions during cancer progression. Traditional two-dimensional (2D) cell culture models are convenient but cannot adequately represent tumor microenvironment histological organization,in vivo3D spatial/cellular context, and physiological relevance. Recently, three-dimensional (3D)in vitrotumor models have been shown to provide an improved platform for incorporating compositional and spatial heterogeneity and to better mimic the biological characteristics of patient tumors to assess drug response. Advances in 3D bioprinting have allowed the creation of more complex models with improved physiologic representation while controlling for reproducibility and accuracy. This review aims to summarize the advantages and challenges of current 3Din vitromodels for evaluating therapy response in breast cancer, with a particular emphasis on 3D bioprinting, and addresses several key issues for future model development as well as their application to other cancers.

Managing type 1 diabetes mellitus (T1DM) presents significant challenges because of the complexity of replicating the microenvironment of pancreatic islets and ensuring the long-term viability and function of transplanted insulin-producing cells (IPCs). This study developed a functional approach that utilizes 3D bioprinting technology to create pore-enriched and pre-vascularized tissue constructs incorporating a pancreatic tissue-derived decellularized extracellular matrix and human-induced pluripotent stem cells (hiPSCs) aimed at enhancing blood glucose regulation in T1DM. We designed a volumetric 3D pancreatic tissue construct that supported the engraftment, survival, and insulin-producing functionality of hiPSC-derived IPCs. The construct's porosity was optimized to enhance IPC delivery efficiency. Additionally, human umbilical vein endothelial cells co-cultured with IPCs in a patterned structure facilitated pre-vascularization, improving construct integration with host tissues and accelerating revascularization post-transplantation. Our results demonstrate high cell viability and sustained insulin production in diabetic rodent models, indicating the constructs' effectiveness in regulating blood glucose levels over an extended period. The findings of this study not only underscore the potential of 3D bioprinting for creating functional tissue constructs for T1DM treatment but also offer efficient cell delivery techniques applicable to other areas of regenerative medicine.

Lung cancer is a serious global health issue that requires the development of patient-specific, lung cancer model for surgical planning to train interventionalists and improve the accuracy of biopsies. Although the emergence of three-dimensional (3D) printing provides a promising solution to create customized models with complicated architectures, current 3D printing methods cannot accurately duplicate anatomical-level lung constructs with tumor(s) which are applicable for hands-on training and procedure planning. To address this issue, an embedded printing strategy is proposed to create respiratory bronchioles, blood vessels, and tumors in a photocurable yield-stress matrix bath. After crosslinking, a patient-specific lung cancer analogous model is produced, which has tunable transparency and mechanical properties to mimic lung parenchyma. This engineered model not only enables the practical training of fine-needle aspiration biopsy but also provides the necessary information, such as coordinates of aspiration, wound depth, and interference with surrounding tissues, for procedure optimization.

The management and treatment of long bone defects are challenging clinical problems. In this study, in order to address the need for load bearing segmental defects, 3D printed cylindrical implants of poly(-caprolactone) (PCL) and nanohydroxyapatite (nHAp) composites were prepared and applied as lateral segments to the femurs of New Zealand white rabbits. The results obtained after 6 weeks of implantation were compared with the autografts. Although the maximum load determined in the 3-point bending tests for the autografts (93±56 N) was higher than the composite implants (57±5 N), histological studies demonstrated similar new bone formation in both test groups. Also, a sizeable callus formation around the autografts and bone ingrowth to the 3D printed implants were observed, and X-ray studies confirmed the formation of the callus. An increase in the bone density around the defect site was detected for both test groups. SEM revealed close interaction between the newly formed bone tissue and the struts of the 3D printed implant. mRUST values, which is an indicator of tissue healing, increased continuously during 6 weeks. In conclusion, 3D printed, 1.5 cm long cylindrical nHAp-PCL implants exhibited excellent bone healing and biomechanical stability in the large lateral segmental bone defects of the rabbits even in a relatively short implantation time as 6 weeks. We believe that these implants could serve as an alternative to autografts in the treatment of long bone defects.

Kidney damage and dysfunction is an emerging health issue worldwide resulting in high morbidity and mortality rates. Numerous renal diseases are recognized to be driven by the immune system. Despite this recognition, the development of targeted therapies has been challenging as knowledge of the underlying mechanism and complex interactions remains insufficient. Recent advancements in the field offer promising avenues for exploring the interplay between renal cells and immune cells and their role in the development of renal inflammation and diseases. This study describes the establishment of a human immunocompetent 3D in vitro co-culture model of the proximal tubule in a high-throughput microfluidic platform that can be used to study renal functionality and inflammatory processes. The model incorporated RPTEC in the top compartment and HUVECs in the bottom compartment cultured under flow and in direct contact with a collagen-I ECM gel resulting in the formation of polarized tubular structures. As an immune component, human primary monocytes of different donors were added to the lumen of the endothelium. Renal inflammation was successfully induced using complement activated serum (CAS) as evident by epithelial morphological changes, increased expression of adhesion molecules, release of pro-inflammatory cytokines, and reduced epithelial viability. Realtime migratory behavior of monocytes showed increased extravasation and migration towards the ECM and Renal compartment upon exposure to CAS with donor-to-donor differences observed. Finally, immune modulatory compounds showed efficacious inhibition of monocyte migration under inflammatory conditions in the microfluidic co-culture model. A successful co-culture model was established and can be applied to study renal functionality in health and disease but also for drug screening due to the compatibility of the platform with automation and relatively high throughput. Overall, the described proximal tubule model has high potential to fill the gap that currently exists to study renal inflammation preclinically. .

Leukemic microenvironment has been recognized as a factor that strongly supports the mechanisms of resistance. Therefore, targeting the microenvironment is currently one of the major directions in drug development and preclinical studies in leukemia. Despite the variety of available leukemia 3D culture models, the reproducible generation of miniaturized leukemic microenvironments, suitable for high-throughput drug testing, has remained a challenge. Here, we use droplet microfluidics to generate tens of thousands of highly monodisperse leukemic-bone marrow microenvironments within minutes. We employ gelatin methacryloyl (GelMA) as a model extracellular matrix (ECM) and tune the concentration of the biopolymer, check the impact of other components of the ECM (hyaluronic acid), cell concentration and the ratio of leukemic cells to bone marrow cells within the microbeads to establish the optimal conditions for microtissue formation. We administer model kinase inhibitor, imatinib, at various concentrations to the encapsulated leukemic microtissues, and, via comparing mono- and co-culture conditions (cancer alone vs cancer-stroma), we find that the stroma-leukemia crosstalk systematically protects the encapsulated cells against the drug-induced cytotoxicity. With that we demonstrate that our system mimics the physiological stroma-dependent protection. We discuss applicability of our model to (i) studying the role of direct- or close-contact interactions between the leukemia and bone marrow cells embedded in microscale 3D ECM on the stroma-mediated protection, and (ii) high-throughput screening of anti-cancer therapeutics in personalized leukemia therapies.

Restoration of disc height and biomechanical function is essential for intervertebral disc degeneration (IDD) treatment. Removing abnormal nucleus pulposus (NP) tissue is an important step to facilitate bony fusion during the healing process. We analyzed publicly available single-cell transcriptome data for human normal and degenerative NP to identify genes associated with NP degeneration. A novel poly(glycolide-co-caprolactone)@polylactide (PLA)-b-aniline pentamer (AP)-b-PLA/chitosan-ϵ-polylysine (PGCL@1PAP/10CSPL) scaffold with good biocompatibility and electroactivity was designed and fabricated as an implant for IDD treatment using 3D printing technology. The PGCL@1PAP/10CSPL scaffold exhibited superior hydrophilicity, mechanical properties, cytocompatibility, and antibacterial activity compared to PGCL. Fibronectin 1 (FN1), identified from single-cell transcriptome analysis, was loaded into the PGCL@1PAP/10CSPL scaffold to accelerate the abnormal NP degeneration.In vitroandin vivoexperiments indicated that the PGCL@1PAP/10CSPL-FN1 scaffold enhanced osteogenic differentiation, promoted angiogenesis, and facilitated the removal of damaged disc tissue. This study introduces a novel implant system with desirable mechanical strength and unique bone-promoting and vascularizing properties for lumbar interbody fusion in IDD treatment.

The lack of the immune component in most of the engineered skin models remains a challenge to study the interplay between different immune and non-immune cell types of the skin. Immunocompetent humanin vitroskin models offer potential advantages in recapitulatingin vivolike behavior which can serve to accelerate translational research and therapeutics development for skin diseases. Here we describe a three-dimensional human full-thickness skin (FTS) equivalent incorporating polarized M1 and M2 macrophages from human peripheral CD14⁺monocytes. This macrophage-incorporated FTS model demonstrates discernible immune responses with physiologically relevant cytokine production and macrophage plasticity under homeostatic and lipopolysaccharide stimulation conditions. M2-incorporated FTS recapitulates skin fibrosis phenotypes with transforming growth factor-β1 treatment as reflected by significant collagen deposition and myofibroblast expression, demonstrating a M2 potentiation effect. In conclusion, we successfully biofabricated an immunocompetent FTS with functional macrophages in a high-throughput (HT) amenable format. This model is the first step towards a HT-assay platform to develop new therapeutics for skin diseases.