A patient-specific lung cancer assembloid model with heterogeneous tumor microenvironments.

Cancer models play critical roles in basic cancer research and precision medicine. However, current in vitro cancer models are limited by their inability to mimic the three-dimensional architecture and heterogeneous tumor microenvironments (TME) of in vivo tumors. Here, we develop an innovative patient-specific lung cancer assembloid (LCA) model by using droplet microfluidic technology based on a microinjection strategy. This method enables precise manipulation of clinical microsamples and rapid generation of LCAs with good intra-batch consistency in size and cell composition by evenly encapsulating patient tumor-derived TME cells and lung cancer organoids inside microgels. LCAs recapitulate the inter- and intratumoral heterogeneity, TME cellular diversity, and genomic and transcriptomic landscape of their parental tumors. LCA model could reconstruct the functional heterogeneity of cancer-associated fibroblasts and reflect the influence of TME on drug responses compared to cancer organoids. Notably, LCAs accurately replicate the clinical outcomes of patients, suggesting the potential of the LCA model to predict personalized treatments. Collectively, our studies provide a valuable method for precisely fabricating cancer assembloids and a promising LCA model for cancer research and personalized medicine.

Towards the Clinical Translation of 3D PLGA/ß-TCP/Mg Composite Scaffold for Cranial Bone Regeneration.

Recent years have witnessed the rapid development of 3D porous scaffolds with excellent biocompatibility, tunable porosity, and pore interconnectivity, sufficient mechanical strength, controlled biodegradability, and favorable osteogenesis for improved results in cranioplasty. However, clinical translation of these scaffolds has lagged far behind, mainly because of the absence of a series of biological evaluations. Herein, we designed and fabricated a composite 3D porous scaffold composed of poly (lactic-co-glycolic) acid (PLGA), ß-tricalcium phosphate (ß-TCP), and Mg using the low-temperature deposition manufacturing (LDM) technique. The LDM-engineered scaffolds possessed highly porous and interconnected microstructures with a porosity of 63%. Meanwhile, the scaffolds exhibited mechanical properties close to that of cancellous bone, as confirmed by the compression tests. It was also found that the original composition of scaffolds could be maintained throughout the fabrication process. Particularly, two important biologic evaluations designed for non-active medical devices, i.e., local effects after implantation and subchronic systemic toxicity tests, were conducted to evaluate the local and systemic toxicity of the scaffolds. Additionally, the scaffolds exhibited significant higher mRNA levels of osteogenic genes compared to control scaffolds, as confirmed by an in vitro osteogenic differentiation test of MC3T3-E1 cells. Finally, we demonstrated the improved cranial bone regeneration performance of the scaffolds in a rabbit model. We envision that our investigation could pave the way for translating the LDM-engineered composite scaffolds into clinical products for cranial bone regeneration.

Creating a semi-opened micro-cavity ovary through sacrificial microspheres as an in vitro model for discovering the potential effect of ovarian toxic agents.

The bio-engineered ovary is an essential technology for treating female infertility. Especially the development of relevant in vitro models could be a critical step in a drug study. Herein, we develop a semi-opened culturing system (SOCS) strategy that maintains a 3D structure of follicles during the culture. Based on the SOCS, we further developed micro-cavity ovary (MCO) with mouse follicles by the microsphere-templated technique, where sacrificial gelatin microspheres were mixed with photo-crosslinkable gelatin methacryloyl (GelMA) to engineer a micro-cavity niche for follicle growth. The semi-opened MCO could support the follicle growing to the antral stage, secreting hormones, and ovulating cumulus-oocyte complex out of the MCO without extra manipulation. The MCO-ovulated oocyte exhibits a highly similar transcriptome to the in vivo counterpart (correlation of 0.97) and can be fertilized. Moreover, we found that a high ROS level could affect the cumulus expansion, which may result in anovulation disorder. The damage could be rescued by melatonin, but the end of cumulus expansion was 3h earlier than anticipation, validating that MCO has the potential for investigating ovarian toxic agents in vitro. We provide a novel approach for building an in vitro ovarian model to recapitulate ovarian functions and test chemical toxicity, suggesting it has the potential for clinical research in the future.

Biofabrication of poly(l-lactide-co-ε-caprolactone)/silk fibroin scaffold for the application as superb anti-calcification tissue engineered prosthetic valve.

In this study, electrospun scaffolds were fabricated by blending poly(l-lactide-co-ε-caprolactone) (PLCL) and silk fibroin (SF) with different ratios, and further the feasibility of electrospun PLCL/SF scaffolds were evaluated for application of tissue engineered heart valve (TEHV). Scanning electron microscopy (SEM) results showed that the surface of PLCL/SF electrospun scaffolds was smooth and uniform while the mechanical properties were appropriate as valve prosthesis. In vitro cytocompatibility evaluation results demonstrated that all of the PLCL/SF electrospun scaffolds were cytocompatible and valvular interstitial cells (VICs) cultured on PLCL/SF scaffolds of 80/20 & 70/30 ratios exhibited the best cytocompatibility. The in vitro osteogenic differentiation of VICs including alkaline phosphatase (ALP) activity and quantitative polymerase chain reaction (qPCR) assays indicated that PLCL/SF scaffolds of 80/20 & 90/10 ratios behaved better anti-calcification ability. In the in vivo calcification evaluation model of rat subdermal implantation, PLCL/SF scaffolds of 80/20 & 90/10 ratios presented better anti-calcification ability, which was consistent with the in vitro results. Moreover, PLCL/SF scaffolds of 80/20 & 70/30 ratios showed significantly enhanced cell infiltration and M2 macrophage with higher CD206+/CD68+ ratio. Collectively, our data demonstrated that electrospun scaffolds with the PLCL/SF ratio of 80/20 hold great potential as TEHV materials.

MWCNT-mediated combinatorial photothermal ablation and chemo-immunotherapy strategy for the treatment of melanoma.

Melanoma, the most aggressive skin cancer with a high metastatic index, causes almost 90% of skin cancer mortality. Currently available conservative therapies, including chemotherapy, radiotherapy and immunotherapy, have shown little effect against metastatic melanoma, leading to a very poor prognosis. The present study was aimed at developing a more efficient therapeutic strategy by combining MWCNT mediated photothermal ablation with both chemotherapy and immunotherapy. For this purpose, DOX and CpG were loaded onto MWCNTs via physical adhesion. The diameters of the resultant MWCNT-CpG and MWCNT-DOX were 197.3 ± 5.45 nm and 263.8 ± 7.36 nm, with zeta potentials of -48 ± 4.93 mV and 58 ± 2.42 mV, respectively. Loading with either CpG or DOX significantly enhanced the water dispersibility of the MWCNTs and showed no obvious impact on the physical structure of the MWCNTs. MWCNT loading facilitated the uptake of CpG by bone marrow derived dendritic cells (BMDCs), as well as the maturation of BMDCs. Intratumoral injection of MWCNT-DOX and MWCNT-CpG with subsequent NIR irradiation resulted in a significant delay in tumor progression in melanoma bearing mice, along with an increased number of CD4+ and CD8+ T cells in the spleen, draining lymph nodes and tumor tissues. The regimen promoted TAM shifting from M2 to M1 while decreasing the number of Treg cells in the tumor microenvironment, which probably contributed to the enhanced anti-tumor efficacy of the regimen. Hopefully, the invented strategy might find potential applications for the therapy of melanoma in the future.

Curcumin-crosslinked acellular bovine pericardium for the application of calcification inhibition heart valves.

Glutaraldehyde (GA) crosslinked bovine or porcine pericardium tissues exhibit high cell toxicity and calcification in the construction of bioprosthetic valves, which accelerate the failure of valve leaflets and motivate the exploration for alternatives. Polyphenols, including curcumin, procyanidin and quercetin, etc, have showed great calcification inhibition potential in crosslinking collagen and elastin scaffolds. Herein, we developed an innovative phenolic fixing technique by using curcumin as the crosslinking reagent for valvular materials. X-ray photoelectron spectroscopy and Fourier transform infrared spectrometry assessments confirmed the hydrogen bond between curcumin and acellular bovine pericardium. Importantly, the calcification inhibition capability of the curcumin-crosslinked bovine pericardium was proved by the dramatically reduced Ca2+ content in the curcumin-fixed group in in vitro assay, a juvenile rat subcutaneous implants model, as well as an osteogenic differentiation model. In addition, the results showed that the curcumin-fixed bovine pericardium exhibited better performance in the areas of mechanical performance, hemocompatibility and cytocompatibility, in comparison with the GA group and the commercialized product. In summary, we demonstrated that curcumin was a feasible crosslinking reagent to fix acellular bovine pericardium, which showed great potential for biomedical applications, particularly in cardiovascular biomaterials with calcification inhibition capacity.

Epoxy Chitosan-Crosslinked Acellular Bovine Pericardium with Improved Anti-calcification and Biological Properties.

Glutaraldehyde (GA) was conventionally used to crosslink bovine pericardium to prepare bioprosthetic heart valves (BHVs), which usually fail within 10 years because of valve deterioration and calcification. To overcome the high cytotoxicity and severe calcification of GA-crosslinked BHVs, a quaternary ammonium salt of epoxy chitosan (epoxy group-modified 3-chlorine-2-hydroxypropyl trimethyl chitosan, abbreviated as "eHTCC") was developed to modify the acellular bovine pericardium to substitute GA and improve its anti-calcification and biocompatible properties. Mechanical test, enzymatic stability test, blood compatibility assay, and cytocompatibility assay were used to investigate its mechanical property and biocompatibility. The anti-calcification effect of the eHTCC-modified bovine pericardium (eHTCC-BP) was assessed by in vitro assay and rat subcutaneous implantation assay. The results showed that eHTCC-BP could improve the mechanical properties and anti-enzymolysis ability of BP, as well as retain the original three-dimensional structure, compared with the uncrosslinked-BP group. Moreover, the in vivo calcification level of the eHTCC-BP group was much lower than that of the GA-BP group, which was 5.1% (2 weeks), 2.3% (4 weeks), and 0.8% (8 weeks) of the GA-BP group. In summary, this study demonstrated that eHTCC could be a potential crosslinking agent for the extracellular matrix for its favorable crosslinking effects, anti-enzymolysis, anti-calcification, and biocompatibility.

Swim Bladder as a Novel Biomaterial for Cardiovascular Materials with Anti-Calcification Properties.

Calcification is a major cause of cardiovascular materials failure and deterioration, which leads to the restriction of their wide application. To develop new materials with anti-calcification capability is an urgent clinical requirement. Herein, a natural material derived from swim bladders as one promising candidate is introduced, which is prepared by decellularization and glutaraldehyde (GA) crosslinking. Data show that the swim bladder is mainly composed of collagen I, glycosaminoglycan (GAG), and elastin, especially rich in elastin, in accordance with higher elastic modulus in comparison to bovine pericardium. Moreover, the calcification of this material is proved dramatically lower than that of bovine pericardium by in vitro calcification assessments and in vivo assay using a rat subcutaneous implantation model. Meanwhile, good cytocompatibility, hemocompatibility, and enzymatic stability are demonstrated by in vitro assays. Further, a small diameter vascular graft using this material is successfully developed by rolling method and in situ implantation assay using a rat abdominal artery replacement model shows great performances in the aspect of higher patency and lower calcification. Taken together, these superior properties of swim bladder-derived material in anti-calcification, proper mechanical strength and stability, and excellent hemocompatibility and cytocompatibility endow it a great candidate as cardiovascular biomaterials.

Bio-orthogonal click reaction-enabled highly specific in situ cellularization of tissue engineering scaffolds.

Tissue engineering generally utilizes natural or synthetic scaffolds to repair or replace damaged tissues. However, due to the lack of guidance of biological signals, most of the implanted scaffolds have always suffered from poor in vivo cellularization. Herein, we demonstrate a bio-orthogonal reaction-based strategy to realize in situ specific and fast cellularization of tissue engineering scaffold. DBCO-modified PCL-PEG (PCL-PEG-DBCO) polymer was synthesized and then fabricated into PCL-PEG-DBCO film through electrospinning. Meanwhile, azide-labeled macrophages (N3 (+) macrophages) were obtained through metabolic glycoengineering. Through a series of in vitro dynamic and in vivo characterization, DBCO-modified films were noted to dramatically increase the selective capture efficiency and survival rate of N3 (+) cells. Additionally, there is negligible influence of covalent conjugation on cell viability and proliferation, indicating the feasibility of the bio-orthogonal click reaction-based tissue engineering strategy. Overall, this work shows the advantages of an in situ bio-orthogonal click reaction in realizing highly specific, efficient, and long-lasting scaffold cellularization. We anticipate that this general strategy would be widely applicable and useful in tissue engineering and regenerative medicine in the near future.

The surrounding tissue contributes to smooth muscle cells' regeneration and vascularization of small diameter vascular grafts.

Small diameter vascular grafts have been promising substitutes for bypass surgery to treat cardiovascular disease. However, no ideal product is available in the clinic. In order to design improved, next generation vascular grafts, it is essential to understand the cellular and molecular mechanisms underlying tissue regeneration after vascular graft implantation. Two diverse microenvironments, circulating blood and the surrounding tissue, are involved in the regeneration process after vascular graft implantation in situ. However, their regenerative functions are not completely understood. To elucidate their roles in regeneration, we used electrospinning to fabricate four types of tubular scaffolds with a structure consisting of a microfiber layer (fiber diameter ∼ 6 µm) and a nanofiber layer (fiber diameter < 1 µm): microfiber scaffold, nanofiber scaffold, outer microfiber bilayer scaffold and inner microfiber bilayer scaffold. In the outer microfiber scaffold, cells from the surrounding tissue were allowed into the scaffold but not cells from the circulating blood while it was opposite in the inner microfiber scaffold. The processes of endothelium formation, smooth muscle cell regeneration, neo-tissue formation and vascularization of these scaffolds were analyzed with a rat left common carotid artery replacement model. Our data showed that smooth muscle cells' regeneration and vascularization were different among the four types of scaffolds. The thickest neo-tissue and α-SMA+ cell layers were detected in the microfiber scaffold group while the thinnest in the nanofiber scaffold group, and thicker neo-tissue and α-SMA+ cell layers were found in the outer microfiber bilayer scaffold group compared to the inner microfiber bilayer scaffold group. In addition, vascularization in the outer microfiber bilayer scaffold group and microfiber group was dramatically better than the inner microfiber bilayer scaffold group and the nanofiber group. Furthermore, we demonstrated that the regenerated SMCs were associated with the CD206+ macrophages in the graft wall. In all, the microfiber scaffold showed the best neo-tissue regeneration in vivo. These results indicate that the surrounding tissue contributes more to vascular regeneration than circulating blood. This finding gives a significant design clue that modulating the vascular surrounding tissue will be an alternative strategy for designing advanced and feasible small diameter vascular grafts.

Nonglutaraldehyde Fixation for off the Shelf Decellularized Bovine Pericardium in Anticalcification Cardiac Valve Applications.

In valvular replacement surgery, especially in the construction of bioprosthetic valves with decellularized pericardial xenograft, glutaraldehyde (GA) is routinely utilized as the golden standard reagent to fix bovine or porcine pericardial tissues. However, the apparent defects of GA, including cytotoxicity and calcification, increase the probability of leaflet failure and motivate the exploration for alternatives. Thus, the aim of this study is to develop nonglutaraldehyde combined-cross-linking reagents composed of alginate-EDC/NHS (Alg) or oxidized alginate-EDC/NHS (Alg-CHO) as substitute for GA, which is confirmed to be less toxic and more biocompatible. Evaluations of the fixed acellular bovine pericardial tissues included mechanical performance, thermodynamics/enzymatic/in vivo stability tests, blood compatibility assay, cytocompatibility assay, in vitro anticalcification, and in vivo anticalcification assay by subcutaneous implantation in juvenile Wistar rats. The data revealed that the tissues fixed with the combined cross-linking reagents were superior to GA control and commercially available Sino product in terms of better in vitro hemocompatibility and cytocompatibility, lower calcification levels, better thermodynamics stability, and better regenerative capacity in subcutaneous implants, while the mechanical strength and in vivo stability were comparable. Considering all above performances, it indicated that both Alg and Alg-CHO are appropriate to replace GA as the cross-linkers for biological tissue, particularly as a nonglutaraldehyde fixation for off the shelf decellularized bovine pericardial tissue in the anticalcification cardiac valve applications. Nevertheless, studies on the long-term durability and calcification-resistance capacity in large animal model are further needed.

Thermal Stability of Metal-Organic Frameworks and Encapsulation of CuO Nanocrystals for Highly Active Catalysis.

We report an aerosol-based approach to study the thermal stability of metal-organic frameworks (MOFs) for gas-phase synthesis of MOF-based hybrid nanostructures used for highly active catalysis. Temperature-programmed electrospray-differential mobility analysis (TP-ES-DMA) provides the characterization of temperature-dependent morphological change directly in the gas phase, and the results are shown to be highly correlated with the structural thermal stability of MOFs determined by the traditional measurements of porosity and crystallinity. The results show that MOFs underwent thermal decomposition via simultaneous disassembly and deaggregation. Trimeric Cr-based MIL-88B-NH2 exhibited a higher temperature of decomposition ( Td), 350 °C, than trimeric Fe-based MIL-88B-NH2, 250 °C. For UiO-66, a significant decrease of Td by ≈100 °C was observed by using amine-functionalized ligands in the MOF structure. Copper oxide nanocrystals were successfully encapsulated in the UiO-66 crystal (Cu xO@UiO-66) by using a gas-phase evaporation-induced self-assembly approach followed by a suitable thermal treatment below Td (i.e., determined by TP-ES-DMA). Cu xO@UiO-66 demonstrated a very high catalytic activity and stability to CO oxidation, showing at least a 3-time increase in CO conversion compared to the bare CuO nanoparticle samples. The study demonstrates a prototype methodology (1) to determine structural thermal stability of MOFs using a gas-phase electrophoretic method (TP-ES-DMA) and (2) to gas-phase synthesize CuO nanocrystals encapsulated in MOFs.