Curcumin nanoparticles combined with 3D printed bionic tumor models for breast cancer treatment.

Compared with conventional therapeutic approaches, nanomedicines are attracting a growing interest due to their better targeting ability, higher delivery efficiency, and good water solubility. However, conventional drug efficacy assessment methods are based on a two-dimensional (2D) culture approach of single cells to obtainin vitrotherapeutic effects, which may not be representative of actual tumors. Based on the above considerations, the three-dimensional (3D) cell culture models became a better choice since they can increase the complexity ofin vitrosystems and provide a biomimetic microenvironment that is closer to thein vivonative than 2D cultures. In our study, curcumin nanoparticle (CurNPs) with good water solubility and good tumor therapeutic effects were prepared by combining polymeric non-ionic surfactant (Pluronic F127) with curcumin. The hybrid scaffolds based on nano-clay, sodium alginate, and gelatin were also prepared, which showed good printability and excellent biocompatibility. We then studied the therapeutic effects of CurNPs on metastatic breast cancer using a 3D tumor model fabricated with scaffold-bound metastatic breast cancer (MDA-MB-231) cells. It was showed that the 3D cell model presented better cell proliferation effect while compared with 2D version. Additionally, there was good enhanced permeability and retention effect when CurNPs entered with better accumulate in 3D cell 'tumor' sites which represented more realistic response of a more real tumor treatment effect for breast cancer cells. Our study indicated that the combinational of nanomaterials with 3D cell 'tumor' models provided an alternative and better platform for drug screening and has great potential be used as safe and effective treatment screening for breast cancer.

Gelatin/sodium alginate hydrogel-coated decellularized porcine coronary artery to construct bilayer tissue engineered blood vessels.

Cardiovascular diseases and vascular trauma can be commonly found in the population. Scholars worldwide hope to develop small-diameter vascular grafts that can replace autologous vessels for clinical use. Decellularized blood vessels can retain the original morphology, structure, and physical properties of blood vessels, which is conducive to cell growth, proliferation, and differentiation. In this study, porcine coronary arteries (PCAs) were decellularized to prepare decellularized porcine coronary artery (DPCA), and bilayer hybrid scaffolds were prepared by coating gelatin and sodium alginate mixed hydrogel of seven different proportions and combined with mouse fibroblasts (L929 cells) to study the construction of tissue engineering vessels in vitro. The obtained bilayer hybrid scaffolds were 3-7 cm in length, 5 mm in external diameter, and 1 mm in average wall thickness. All seven bilayer hybrid scaffolds showed good biocompatibility after cell inoculation. Compared with 2D culture, cells on 3D scaffolds grew relatively slowly in the first 4 days, and the number of cells proliferated rapidly at 7 days. In the same culture days, different concentrations of hydrogel also had an impact on cell proliferation. With the increase of hydrogel content, cells on the 3D scaffold formed cell colonies faster. The results showed that the scaffold had good biocompatibility and could meet the needs of artificial blood vessel construction.

Decellularized Pig Kidney with a Micro-Nano Secondary Structure Contributes to Tumor Progression in 3D Tumor Model.

In spite of many anti-cancer drugs utilized in clinical treatment, cancer is still one of the diseases with the highest morbidity and mortality worldwide, owing to the complexity and heterogeneity of the tumor microenvironment. Compared with conventional 2D tumor models, 3D scaffolds could provide structures and a microenvironment which stimulate native tumor tissues more accurately. The extracellular matrix (ECM) is the main component of the cell in the microenvironment that is mainly composed of three-dimensional nanofibers, which can form nanoscale fiber networks, while the decellularized extracellular matrix (dECM) has been widely applied to engineered scaffolds. In this study, pig kidney was used as the source material to prepare dECM scaffolds. A chemical crosslinking method was used to improve the mechanical properties and other physical characteristics of the decellularized pig kidney-derived scaffold. Furthermore, a human breast cancer cell line (MCF-7) was used to further investigate the biocompatibility of the scaffold to fabricate a tumor model. The results showed that the existence of nanostructures in the scaffold plays an important role in cell adhesion, proliferation, and differentiation. Therefore, the pig kidney-derived matrix scaffold prepared by decellularization could provide more cell attachment sites, which is conducive to cell adhesion and proliferation, physiological activities, and tumor model construction.

Dynamic process enhancement on chitosan/gelatin/nano-hydroxyapatite-bone derived multilayer scaffold for osteochondral tissue repair.

Accurate repair of osteochondral defects is a great challenge due to the complex structure of osteochondral defects. The current study aims to prepare a biomimetic osteochondral gradient scaffold based on chitosan, gelatin and nano-hydroxyapatite and bone-derived materials for repair cartilage defects. Hereon, the chitosan/gelatin/nano-hydroxyapatite multilayer scaffold with complex hierarchical structure using iterative hierarchical method is prepared to simulate the structure of natural cartilage. And porcine femur in distal metaphysis was treated by chemical decellularization, deproteinization, decalcification, and degreasing to obtain the bone-derived scaffold simulating subchondral bone layer. We also used a spinner bioreactor for the in vitro modeling of the microenvironment which can promote adipose mesenchymal stem cell (ADSCs) recruitment into the chitosan/gelatin/nano-hydroxyapatite-bone derived multilayer scaffold under physiological flow conditions. Biological experiments have shown that osteochondral layered materials can induce ADSCs to differentiate into chondrocytes and osteoblasts and exhibit chondrogenic and osteogenic phenotypes similar to natural tissues, respectively. Furthermore, the expression both of the chondrogenic gene (Col II, ACAN, and Sox9) and osteogenic gene (Runx2, OCN and Col I) of ADSCs differentiated by mechanical stimulation were increased. These results indicate that osteochondral materials and dynamic environment are the key factors to improve cell regulation or signal molecule transfer function, and provide a feasible plan for osteochondral regeneration for future medical services.

A biological functional hybrid scaffold based on decellularized extracellular matrix/gelatin/chitosan with high biocompatibility and antibacterial activity for skin tissue engineering.

Nowadays, decellularized extracellular matrix (dECM) has received widespread attention due to its diversity in providing the unique structural and functional components to support cell growth, and finding material with good biocompatibility and anti-infection capability for skin tissue engineering is still a challenge. In this study, a novel dECM/Gel/CS scaffold with appropriate mechanical strength, good antibacterial activity and high biocompatibility was prepared using a one-pot method. The results showed that the immune components such as cells and DNA (about 98.1%) were successfully removed from the porcine skin tissue. The dECM/Gel/CS scaffolds exhibited an interconnected pore structure and had a high porosity (>90%) to promote cell growth. Moreover, the appropriate elastic modulus (≥482.17 kPa) and degradability (≥80.04% for 15 days) of the scaffolds offered stout "houses" for cell proliferation and suitable degradation rate to match the new tissue formation in skin tissue engineering. Furthermore, the addition of chitosan endowed the scaffold with good antibacterial activity, water and protein absorption capacity to avoid wound infection, and maintain the moisture and nutrition balance. In vitro cytocompatibility studies showed that the presence of dECM effectively enhanced the cell proliferation. Overall, the advanced dECM/Gel/CS scaffold has considerable potential to be applied in skin tissue engineering.

Evaluation of anti-tumor effects of crocin on a novel 3D tissue-engineered tumor model based on sodium alginate/gelatin microbead.

Crocin, as one of the biologically active components of saffron, has anti-inflammatory, anti-oxidant, anti-depressant and auxiliary anti-tumor effects. Studies have shown that crocin could promote breast cancer cell apoptosis. However, conventional methods are mainly based on two-dimensional (2D) cell culture models, which are difficult to reproduce the tumor environment in vivo due to space constraints. In this study, we prepared a three-dimensional (3D) cell model in vitro based on sodium alginate/gelatin to evaluate the inhibitory effect of crocin on MCF-7 cells, which could bridge the gap in crocin drug evaluation between 2D and 3D cell model in vitro. Different from the 2D culture, the cells were found to aggregate in a spherical shape in the 3D microgel beads. And the CCK-8 assay showed that the growth of MCF-7 cells exposed to crocin was inhibited in a time-related and concentration-related manner. Compared with 2D culture (IC50 that MCF-7 cells treated with crocin at 24 h, 48 h, 72 h: 3.68, 2.55 and 1.53 mg/mL, respectively), the IC50 value of 3D culture (IC50 that MCF-7 cells treated with crocin at 24 h, 48 h, 72 h: 10.12, 6.89 and 6.64 mg/mL, respectively) was significantly increased by 2.77, 2.70, 4.34 times, respectively. Besides, live/dead staining and scanning electron microscope (SEM) revealed that the 2D cultured cells shrank and ruptured after crocin treatment, and the number of living cells was considerably reduced; the size of the cell colonies in the 3D microgel beads decreased.

Evaluation of inhibitory effects of geniposide on a tumor model of human breast cancer based on 3D printed Cs/Gel hybrid scaffold.

Traditional Chinese medicine therapy, which can serve as adjuvant therapy for cancer treatment, has no obvious side effects on the human body. Geniposide (GEN), one of the main iridoid glycosides in gardenia fruit, has been widely reported to have anti-cancer effects. In this study, we aimed to inspect whether GEN could inhibit proliferation and promote the apoptosis of human breast cancer cells (MCF-7). In order to better predict the efficacy of GEN, we have prepared the Cs/Gel composite scaffolds by 3D printing technology to mimic the MCF-7 cell growth microenvironment. The prepared Cs/Gel scaffold has good mechanical properties and biocompatibility, which can provide a more accurate platform for drug screening. The semi-inhibitory concentration (IC50) evaluated by CCK-8 assay was 16.06 mg/mL (24 h), 14.85 mg/mL (48 h), and 13.14 mg/mL (72 h). After exposed to GEN for 48 h, the cancer cell survival rate reduced from 69.15 ± 2.86% (13 mg/mL) to 20.97 ± 3.24% (16 mg/mL). Although the inhibitory effect was weaker in the 3D culture system, it also managed to inhibit cell proliferation and induce cell apoptosis. Besides, Live/Dead staining, Hematoxylin-Eosin (H&E) staining and SEM evaluation were also conducted to estimate the anti-cancer effect of GEN in 2D and 3D cultures. The results indicate that GEN has an anti-cancer effect based on a time- and dose-dependent manner.

Toxicological evaluation of ionic liquid in a biological functional tissue construct model based on nano-hydroxyapatite/chitosan/gelatin hybrid scaffolds.

The application of ionic liquid is attracting more attentions as green replacement for volatile organic solvents. However, the toxic effects and risks of ionic liquid in different biological systems for human health and environment are poorly evaluated. Among all ionic liquids, 1-ethyl-3-methylimidazolium diethylphosphate ([Emim]DEP-type) ionic liquid is still at the early phase of development, and its toxicity remains unclear. In this study, we fabricated a 3D biological functional tissue construct model based on nano-hydroxyapatite, chitosan and gelatin hybrid scaffold and evaluated its toxic effects of [Emim]DEP-type ionic liquid. As a control group, the examination of ionic liquid's toxic effects on the pre-osteoblast cell line (MC3T3-E1) was detected in 2D cultures. The MTT assay showed that [Emim]DEP-type ionic liquid inhibited the proliferation of cells on both 2D cultures and 3D tissue constructs. This effect was correlated with culturing time and concentration, while the IC50 on 3D scaffolds (12,566, 9015, 7896 µg/mL, at 24 h, 48 h and 72 h, respectively) was found significantly higher compared to 2D cultures (3959, 2226, 1884 µg/mL). Flow cytometry analysis and scanning electron microscope demonstrated that when [Emim]DEP-type ionic liquid acted on MC3T3-E1 cells for 48 h, the shape of 2D cells shrank, together with decreased surface adhesion.

Characterization and anti-tumor bioactivity of astragalus polysaccharides by immunomodulation.

Astragalus polysaccharide (APS) has attracted growing interests in the field of anti-cancer by direct killing effect and improving immune function. In this study, the structure and composition of APS was determined, following the evaluation of in vitro and in vivo anti-tumor activity of APS targeted macrophages and host immune system based on immunoregulated strategy. The results indicated that APS had no direct cytotoxicity against 4T1 cells, but APS mediated macrophages could significantly inhibit the growth of 4T1 cells by the induction of cell cycle arrest (G2 phase) and cell apoptosis. APS mediated macrophages promoted the apoptosis of 4T1 cells mainly through the mitochondrial apoptosis pathway. The in vivo findings demonstrated that APS could markedly improve the thymus index and spleen index, and restore the structure of the damaged thymus and spleen tissue. APS could significantly enhance the proliferation of spleen lymphocytes and increase phagocytosis of peritoneal macrophages in mice. Furthermore, APS was capable of up-regulating the expression of IL-2, TNF-α and IFN-γ in peripheral blood. APS combined with 5-FU could improve the anti-tumor effect accompanied by the immunosuppressive alleviation of 5-FU on immune system, which may be suitable as an immune adjuvant for chemotherapy.

In Vitro Fabrication and Biocompatibility Assay of a Biomimetic Osteoblastic Niche.

A novel HAp-CS/Gel biomimetic osteoblastic niche was fabricated by freeze-drying, and its mechanical strength and biocompatibility were characterized. HAp-CS/Gel scaffolds in various ratios of 100:5, 100:10, and 100:20 (CS/Gel to HAp) were prepared by freeze-drying prior to chemical cross-linking followed by infrared spectrum analysis, EDS, FITR, SEM, fluorescence microscopy, MTT, and ALP experiments. Results from the infrared spectrum analysis showed that HAp doping remained the surface morphology and the architecture of scaffold with interconnected pores in the size range of 135 to 150 µm. The HAp doping ratio of 100:20 was found to be optimal based on its high porosity of 90%, better water uptake folds of 19.1. In addition, EDS and FITR analyses demonstrated that HAps were uniformly distributed on the surface of a scaffold with aggregates and particles, which has sufficient roughness for cell attachment and proliferation of osteoblasts. Under SEM and fluorescent microscopy, osteoblasts seeded onto the scaffold showed evenly distributed viable cells, which is believed to form a biomimetic niche. In the present study, we further demonstrate that osteoblasts can maintain their function and grow well on the scaffold through MTT and ALP tests. Thus, the scaffold has favorable physical properties and biocompatibility to support the proliferation and differentiation of osteoblasts and further to support the constructs of biomimetic osteoblastic niche.

Fabrication and detection of tissue engineered bone aggregates based on encapsulated human ADSCs within hybrid calcium alginate/bone powder gel-beads in a spinner flask.

Traditional treatment for bone diseases limits their clinical application due to undesirable host immune rejection, limited donator sources and severe pain and suffering for patients. Bone tissue engineering therefore is expected to be a more effective way in treating bone diseases. In the present study, hybrid calcium alginate/bone powder gel-beads with a uniform size distribution, good biocompatibility and osteoinductive capability, were prepared to be used as an in-vitro niche-like matrix. The beads were optimized using 2.5% (w/v) sodium alginate solution, 4.5% (w/v) CaCl2 solution and 5.0mg/mL bone powder using an easy-to-use method. Human ADSCs were cultured and induced into chondrocytes and osteoblasts, respectively. The cells were characterized by histological staining showing the ADSCs were able to maintain their characteristic morphology with multipotent differentiation ability. ADSCs at density of 5 × 10(6)cells/mL were encapsulated into the gel-beads aiming to explore cell expansion under different conditions and the osteogenic induction of ADSCs was verified by specific staining. Results demonstrated that the encapsulated ADSCs expanded 5.6 folds in 10 days under dynamic condition via spinner flask, and were able to differentiate into osteoblasts (OBs) with extensive mineralized nodules forming the bone aggregates over 3 weeks postosteogenic induction. In summary, hybrid gel-beads encapsulating ADSCs are proved to be feasible as a new method to fabricate tissue engineered bone aggregation with potential to treat skeletal injury in the near future.

Three-dimensional dynamic fabrication of engineered cartilage based on chitosan/gelatin hybrid hydrogel scaffold in a spinner flask with a special designed steel frame.

Cartilage transplantation using in vitro tissue engineered cartilage is considered a promising treatment for articular cartilage defects. In this study, we assessed the advantages of adipose derived stem cells (ADSCs) combined with chitosan/gelatin hybrid hydrogel scaffolds, which acted as a cartilage biomimetic scaffold, to fabricate a tissue engineered cartilage dynamically in vitro and compared this with traditional static culture. Physical properties of the hydrogel scaffolds were evaluated and ADSCs were inoculated into the hydrogel at a density of 1×10(7) cells/mL and cultured in a spinner flask with a special designed steel framework and feed with chondrogenic inductive media for two weeks. The results showed that the average pore size, porosity, swelling rate and elasticity modulus of hybrid scaffolds with good biocompatibility were 118.25±19.51 µm, 82.60±2.34%, 361.28±0.47% and 61.2±0.16 kPa, respectively. ADSCs grew well in chitosan/gelatin hybrid scaffold and successfully differentiated into chondrocytes, showing that the scaffolds were suitable for tissue engineering applications in cartilage regeneration. Induced cells cultivated in a dynamic spinner flask with a special designed steel frame expressed more proteoglycans and the cell distribution was much more uniform with the scaffold being filled mostly with extracellular matrix produced by cells. A spinner flask with framework promoted proliferation and chondrogenic differentiation of ADSCs within chitosan/gelatin hybrid scaffolds and accelerated dynamic fabrication of cell-hydrogel constructs, which could be a selective and good method to construct tissue engineered cartilage in vitro.

Fabrication and Cell Responsive Behavior of Macroporous PLLA/Gelatin Composite Scaffold with Hierarchical Micro-Nano Pore Structure.

Scaffolds providing a 3D environment which can effectively promote the adhesion, proliferation and differentiation of cells are crucial to tissue regeneration. In this study, the poly-l-lactic acid (PLLA) scaffold with hierarchical pore structural was fabricated via two-step thermally induced phase separation (TIPS). To mimic both physical architecture and chemical composite of natural bone extracellular matrix (ECM), gelatin fibers were introduced into the pores of PLLA scaffolds and formed 3D network structure via TIPS. Human adipose tissue-derived stem cells (ADSCs) were harvested and seeded into PLLA/gel hybrid scaffolds and cultured in vitro for biocompatibility assay. The surface morphology, porosity and compressive modulus of scaffolds were characterized by scanning electron microscopy (SEM), density analysis and compression test respectively. The results showed that hybrid scaffolds had high porosity (91.62%), a good compressive modulus (2.79 ± 0.20 MPa), nanometer fibers (diameter around 186.39~354.30 nm) and different grades of pore size from 7.41 ± 2.64 nm to 387.94 ± 102.48 nm. The scaffolds with mild hydrolysis by NaOH were modified by 1-ethyl-3-(3-dimethyl ami-nopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS). Gelatin was performed onto PLLA scaffold via TIPS aiming at enhancement cell-material interaction. In comparison with PLLA scaffold, the PLLA/gel scaffold had better biological performance and the mechanical properties because the gelatin fibers homogeneously distributed in each pore of PLLA scaffold and formed 3D network structure.