Bioprinted, spatially defined breast tumor microenvironment models of intratumoral heterogeneity and drug resistance.

Cellular, extracellular matrix (ECM), and spatial heterogeneity of tumor microenvironments (TMEs) regulate disease progression and treatment efficacy. Developing in vitro models that recapitulate the TME promises to accelerate studies of tumor biology and identify new targets for therapy. Here, we used extrusion-based, multi-nozzle 3D bioprinting to spatially pattern triple-negative MDA-MB-231 breast cancer cells, endothelial cells (ECs), and human mammary cancer-associated fibroblasts (HMCAFs) with biomimetic ECM inks. Bioprinted models captured key features of the spatial architecture of human breast tumors, including varying-sized dense regions of cancer cells and surrounding microvessel-rich stroma. Angiogenesis and ECM stiffening occurred in the stromal area but not the cancer cell-rich (CCR) regions, mimicking pathological changes in patient samples. Transcriptomic analyses revealed upregulation of angiogenesis-related and ECM remodeling-related signatures in the stroma region and identified potential ligand-receptor (LR) mediators of these processes. Breast cancer cells in distinct parts of the bioprinted TME showed differing sensitivities to chemotherapy, highlighting environmentally mediated drug resistance. In summary, our 3D-bioprinted tumor model will act as a platform to discover integrated functions of the TME in cancer biology and therapy.

Biofabrication of Composite Tendon Constructs with the Fibrous Arrangement, High Cell Density, and Enhanced Cell Alignment.

Current tissue-engineered tendons are mostly limited to the replication of fibrous organizations of native tendons, which lack the biomimicry of a densely packed cell arrangement. In this study, composite tendon constructs (CTCs) with fibrous arrangement, high cell density, and enhanced cell alignment were developed by integrating the electrohydrodynamic jet 3D printing (e-jetting) technique and the fabrication of tissue strands (TSs). A tubular polycaprolactone (PCL) scaffold was created using e-jetting, followed by coating a thin layer of alginate. Human mesenchymal stem cells were then microinjected into the PCL scaffolds, aggregated into TSs, and formed CTCs with a core-shell structure. Owing to the presence of TSs, CTCs demonstrated the anatomically relevant cell density and morphology, and cells migrated from the TSs onto e-jetted scaffolds. Also, the mechanical strength of CTCs approached that of native tendons due to the existence of e-jetted scaffolds (Young's modulus: ∼21 MPa, ultimate strength: ∼5 MPa). During the entire culture period, CTCs maintained high survival rates and good structural integrity without the observation of necrotic cores and disintegration of two portions. In addition, CTCs that were cultured with uniaxial cyclic stretching revealed not only the increased expression of tendon-related proteins but also the enhanced cellular orientation. The promising results demonstrated the potential of this novel biofabrication strategy for building tissue-engineered tendon constructs with the proper biological, mechanical, and histological relevance..

Naturally derived highly resilient and adhesive hydrogels with application as surgical adhesive.

The inadequacy of conventional surgical techniques for wound closure and repair in soft and resilient tissues may lead to poor healing outcomes such as local tissue fibrosis and contracture. Therefore, the development of adhesive and resilient hydrogels that can adhere firmly to irregular and dynamic wound interfaces and provide a "tension-free proximity" environment for tissue regeneration has become extremely important. Herein, we describe an integrated modeling-experiment-application strategy for engineering a promising hydrogel-based bioadhesive based on recombinant human collagen (RHC) and catechol-modified hyaluronic acid (HA-Cat). Molecular modeling and simulations were used to verify and explore the hypothesis that RHC and HA-Cat can form an assembly complex through physical interactions. The complex was synergistically crosslinked via a catechol/o-quinone coupling reaction and a carbodiimide coupling reactions, resulting in superior hydrogels with strong adhesion and resilience properties. The application of this bioadhesive to tissue adhesion and wound sealing in vivo was successfully demonstrated, with an optimum collagen index, epidermal thickness, and lowest scar width. Furthermore, subcutaneous implantation demonstrated that the bioadhesive exhibited good biocompatibility and degradability. This newly developed hydrogel may be a highly promising surgical adhesive for medical applications, including wound closure and repair.

A Study on Dual-Response Composite Hydrogels Based on Oriented Nanocellulose.

In nature, many biological tissues are composed of oriented structures, which endow tissues with special properties and functions. Although traditional hydrogels can achieve a high level of biomimetic composition, the orderly arrangement of internal structures remains a challenge. Therefore, it is of great significance to synthesize hydrogels with oriented structures easily and quickly. In this study, we first proposed and demonstrated a fabrication process for producing a well-ordered and dual-responsive cellulose nanofibers + hyaluronic acid methacrylate (CN+HAMA) hydrogels through an extrusion-based three-dimensional (3D) printing process. CN in the CN+HAMA hydrogels are directionally aligned after extrusion due to shear stress. In addition, the synthesized hydrogels exhibited responsive behaviors to both temperature and ultraviolet light. Since the temperature-responsiveness is reversible, the hydrogels can transit between the gelation and solution states while retaining their original qualities. Furthermore, the developed well-oriented CN+HAMA hydrogels induced directional cell growth, paving the way for potential applications in ordered biological soft-tissue repair.

Long-term cultured microvascular networks on chip for tumor vascularization research and drug testing.

The vascular structure of the tumor microenvironment (TME) plays an essential role in the process of metastasis. In vitro microvascular structures that can be maintained for a long time will greatly promote metastasis research. In this study, we constructed a mimicking breast cancer invasion model based on a microfluidic chip platform, and the maintenance time of the self-assembled microvascular networks significantly improved by culturing with fibroblasts (up to 13 days). Using this model, we quantified the invasion ability of breast cancer cells and angiogenesis sprouts caused by cancer cells, and the intravasation behavior of cancer cells was also observed in sprouts. We found that cancer cells could significantly cause angiogenesis by promoting sprouting behaviors of the self-assembled human umbilical vein endothelial cells, which, in turn, promoted the invasion behavior of cancer cells. The drug test results showed that the drug resistance of the widely used anti-cancer drugs 5-Fluorouracil (5-FU) and Doxorubicin (DOX) in the 3D model was higher than that in the 2D model. Meanwhile, we also proved that 5-FU and DOX had the effect of destroying tumor blood vessels. The anti-angiogenic drug Apatinib (VEGFR inhibitor) enhanced the drug effect of DOX on MDA-MB-231 cells, further proving the promoting effect of angiogenesis on the invasion ability of cancer cells. These results indicate that our model is of great value in reconstructing TME and drug testing in vitro.

A Three-Dimensional Co-Culture Model for Rheumatoid Arthritis Pannus Tissue.

Three-dimensional (3D) co-culture models have closer physiological cell composition and behavior than traditional 2D culture. They exhibit pharmacological effects like in vivo responses, and therefore serve as a high-throughput drug screening model to evaluate drug efficacy and safety in vitro. In this study, we created a 3D co-culture environment to mimic pathological characteristics of rheumatoid arthritis (RA) pannus tissue. 3D scaffold was constructed by bioprinting technology with synovial fibroblasts (MH7A), vascular endothelial cells (EA.hy 926) and gelatin/alginate hydrogels. Cell viability was observed during 7-day culture and the proliferation rate of co-culture cells showed a stable increase stage. Cell-cell interactions were evaluated in the 3D printed scaffold and we found that spheroid size increased with time. TNF-α stimulated MH7A and EA.hy 926 in 3D pannus model showed higher vascular endothelial growth factor (VEGF) and angiopoietin (ANG) protein expression over time. For drug validation, methotrexate (MTX) was used to examine inhibition effects of angiogenesis in 3D pannus co-culture model. In conclusion, this 3D co-culture pannus model with biological characteristics may help the development of anti-RA drug research.

[Genetic diversity and phylogeny of soybean bradyrhizobia isolated from south and north region of China].

Studies on genetic diversity and phylogeny of soybean bradyrhizobia isolated from south and north region of China were investigated through 16S rRNA gene PCR RFLP, 16S rRNA gene sequencing and 16S-23S rRNA IGS PCR RFLP assays. Results of 16S rRNA gene PCR RFLP and 16S rRNA gene sequencing analysis reveal that strains tested are ascribed into Bradyrhizobium japonicum and B. elkanii species. B. japonicum is the dominant species and accounts for 91% of strains tested, but B. elkanii just up to 9%, which shows poor genetic diversity. Results of 16S-23S rRNA IGS PCR RFLP assays reveal that strains belonged to B. japonicum can be divided into Group I and Group HIIat the 69 similarity. Group I consists of strains from northern China, and Group 1I consists of strains from southern China, which demonstrates geographical effect on genetic diversity of bradyrhzobia. Results of 16S-23S rRNA IGS PCR RFLP further reflects that strains of Group I and Group II are phylogenetically different with type or representative strains USDA6, USDA110 and USDA122 of B. japonicum.

[Studies on genetic diversity and phylogeny of slow-growing rhizobia isolated from Vigna radiata at main ecotypes of China].

Studies on genetic diversity and phylogeny of slow-growing rhizobia isolated from Vigna radiata at main ecotypes of China were conducted by using 16S rRNA gene PCR-RFLP, 16S rRNA gene sequencing and 16S-23S rRNA IGS PCR-RFLP assays. Results of 16S rRNA gene PCR RFLP analysis reveal that all the strains tested are clustered into three groups at the similarity of 76%. Group I contains 13 slow-growing rhizobia tested including LYG1; Group II consists of 21 strains tested and the type strains of B. japonicum and B. liaoningense, and 10 tested strains isolated from Guangdong, Guangxi and the type strain of B. elkanii compose Group III . The results of 16S-23S rRNA IGS PCR-RFLP show that strains tested could be divided into A and B groups, which could be correspondently subdivided into A I , A II , AIII , BI and B II subgroups at the similarity of 85% . Compared with 16S rRNA PCR-RFLP, IGS RFLP assay show higher resolution, strains and reference strains tested can be divided into 21 IGS RFLP patterns. The strains isolated from Xinjiang, Guangdong and Guangxi regions show obvious geographical effect on genetic diversity.