3D bioprinting of in vitro porous hepatoma models:establishment,evaluation,and anticancer drug testing

Traditional tumor models do not tend to accurately simulate tumor growth in vitro or enable personalized treatment and are particularly unable to discover more beneficial targeted drugs.To address this,this study describes the use of threedimensional(3D)bioprinting technology to construct a 3D model with human hepatocarcinoma SMMC-7721 cells(3DP-7721)by combining gelatin methacrylate(GelMA)and poly(ethylene oxide)(PEO)as two immiscible aqueous phases to form a bioink and innovatively applying fluorescent carbon quantum dots for long-term tracking of cells.The GelMA(10%,mass fraction)and PEO(1.6%,mass fraction)hydrogel with 3:1 volume ratio offered distinct pore-forming characteristics,satisfactorymechanical properties,and biocompatibility for the creation of the 3DP-7721 model.Immunofluorescence analysis and quantitative real-time fluorescence polymerase chain reaction(PCR)were used to evaluate the biological properties of the model.Compared with the two-dimensional culture cell model(2D-7721)and the 3D mixed culture cell model(3DM-7721),3DP-7721 significantly improved the proliferation of cells and expression of tumor-related proteins and genes.Moreover,we evaluated the differences between the three culture models and the effectiveness of antitumor drugs in the three models and discovered that the efficacy of antitumor drugs varied because of significant differences in resistance proteins and genes between the three models.In addition,the comparison of tumor formation in the three models found that the cells cultured by the 3DP-7721 model had strong tumorigenicity in nude mice.Immunohistochemical evaluation of the levels of biochemical indicators related to the formation of solid tumors showed that the 3DP-7721 model group exhibited pathological characteristics of malignant tumors,the generated solid tumors were similar to actual tumors,and the deterioration was higher.This research therefore acts as a foundation for the application of 3DP-7721 models in drug development research.

3D printed-electrospun PCL/hydroxyapatite/MWCNTs scaffolds for the repair of subchondral bone

Osteochondral defect caused by trauma or osteoarthritis exhibits a major challenge in clinical treatment with limited symptomatic effects at present.The regeneration and remodeling of subchondral bone play a positive effect on cartilage regeneration and further promotes the repair of osteochondral defects.Making use of the strengths of each preparation method,the combination of 3D printing and electrospinning is a promising method for designing and constructing multi-scale scaffolds that mimic the complexity and hierarchical structure of subchondral bone at the microscale and nanoscale,respectively.In this study,the 3D printed-electrospun poly(ɛ-caprolactone)/nano-hydroxyapatites/multi-walled carbon nanotubes(PCL/nHA/MWCNTs)scaffolds were successfully constructed by the combination of electrospinning and layer-by-layer 3D printing.The resulting dual-scale scaffold consisted of a dense layer of disordered nanospun fibers and a porous microscale 3D scaffold layer to support and promote the ingrowth of subchondral bone.Herein,the biomimetic PCL/nHA/MWCNTs scaffolds enhanced cell seeding efficiency and allowed for higher cell-cell interactions that supported the adhesion,proliferation,activity,morphology and subsequently improved the osteogenic differentiation of bone marrow mesenchymal stem cells in vitro.Together,this study elucidates that the construction of 3D printed-electrospun PCL/nHA/MWCNTs scaffolds provides an alternative strategy for the regeneration of subchondral bone and lays a foundation for subsequent in vivo studies.

Mesenchymal stem cells loaded on 3D-printed gradient poly(e-caprolactone)/methacrylated alginate composite scaffolds for cartilage tissue engineering

Cartilage has limited self-repair ability due to its avascular,alymphatic and aneural features.The combination of three-dimensional(3D)printing and tissue engineering provides an up-and-coming approach to address this issue.Here,we designed and fabricated a tri-layered(superficial layer(SL),middle layer(ML)and deep layer(DL))stratified scaffold,inspired by the architecture of collagen fibers in native cartilage tissue.The scaffold was composed of 3D printed depth-dependent gradient poly(e-caprolactone)(PCL)impregnated with methacrylated alginate(ALMA),and its morphological analysis and mechanical properties were tested.To prove the feasibility of the composite scaffolds for cartilage regeneration,the viability,proliferation,collagen deposition and chondrogenic differentiation of embedded rat bone marrow mesenchymal stem cells(BMSCs)in the scaffolds were assessed by Live/dead assay,CCK-8,DNA content,cell morphology,immunofluorescence and real-time reverse transcription polymerase chain reaction.BMSCs-loaded gradient PCL/ALMA scaffolds showed excellent cell survival,cell proliferation,cell morphology,collagen II deposition and hopeful chondrogenic differentiation compared with three individual-layer scaffolds.Hence,our study demonstrates the potential use of the gradient PCL/ALMA construct for enhanced cartilage tissue engineering.

Biocompatible chitosan/polyethylene glycol/multi-walled carbon nanotube composite scaffolds for neural tissue engineering

Carbon nanotube(CNT)composite materials are very attractive for use in neural tissue engineering and biosensor coatings.CNT scaffolds are excellent mimics of extracellular matrix due to their hydrophilicity,viscosity,and biocompatibility.CNTs can also impart conductivity to other insulating materials improve mechanical stability guide neuronal cell behavior and trigger axon regeneration.The performance of chitosan(CS)/polyethylene glycol(PEG)composite scaffolds could be optimized by introducing multi-walled CNTs(MWCNTs).CS/PEG/CNT composite scaffolds with CNT content of 1%,3%,and 5%(1%=0.01 g/mL)were prepared by freeze-drying.Their physical and chemical properties and biocompatibility were evaluated.Scanning electron microscopy(SEM)showed that the composite scaffolds had a highly connected porous structure.Transmission electron microscope(TEM)and Raman spectroscopy proved that the CNTs were well dispersed in the CS/PEG matrix and combined with the CS/PEG nanofiber bundles.MWCNTs enhanced the elastic modulus of the scaffold.The porosity of the scaffolds ranged from 83%to 96%.They reached a stable water swelling state within 24 h,and swelling decreased with increasing MWCNT concentration.The electrical conductivity and cell adhesion rate of the scaffolds increased with increasing MWCNT content.Immunofluorescence showed that rat pheochromocytoma(PC12)cells grown in the scaffolds had characteristics similar to nerve cells.We measured changes in the expression of nerve cell markers by quantitative real-time polymerase chain reaction(qRT-PCR),and found that PC12 cells cultured in the scaffolds expressed growth-associated protein 43(GAP43),nerve growth factor receptor(NGFR),and class IIIβ-tubulin(TUBB3)proteins.Preliminary research showed that the prepared CS/PEG/CNT scaffold has good biocompatibility and can be further applied to neural tissue engineering research.