Premetastatic Niche Mimicking Bone-On-A-Chip: A Microfluidic Platform to Study Bone Metastasis in Cancer Patients.

Primary cancer modulates the bone microenvironment to sow the seeds of dormancy and metastasis in tumor cells, leading to multiple organ metastasis and death. In this study, 3D printing and bone-on-a-chip (BOC) are combined to develop a BOC platform that mimics the pre-metastatic niches (PMNs) and facilitates elucidation of the interactions between bone-resident cells and metastatic tumor cells under the influence of primary cancer. Photocrosslinkable gelatin methacrylate (GelMA) is used as a 3D culturing hydrogel to encapsulate cells, and circulate tumor culture medium (CM) adjacent to the hydrogel to verify the critical role of mesenchymal stem cells (MSCs) and osteoclasts (RAW264.7s). Three niches: the dormancy niche, the perivascular niche, and the "vicious cycle" niche, are devised to recapitulate bone metastasis in one chip with high cell viability and excellent nutrient exchange. With respect to tumor dormancy and reactivation, the invadopodia formation of A549 lung cancer cells in communication with MSCs and RAW264.7 via the cortactin pathway is researched. As a proof of concept, the functionality and practicality of the platform are demonstrated by analyzing the invadopodia formation and the influence of various cells, and the establishment of the dynamic niches paves the way to understanding PMN formation and related drug discovery.

Biodegradable photothermal thermosensitive hydrogels treat osteosarcoma by reprogramming macrophages.

Osteosarcoma is one of the most common malignant tumors in children and tends to occur around the knee. Problems such as recurrence and metastasis are the outcomes of traditional treatment methods. One of the reasons for these issues is the infiltration of tumor-associated macrophages (TAMs) in the tumor microenvironment (TME). Photothermal immunotherapy has emerged as one of the most potent approaches for cancer treatment. In this study, we designed a biodegradable, injectable, and photothermal hydrogel that functions to reprogram TAMs into classically activated macrophages (M1) based on hydroxypropyl chitin (HPCH), tannic acid and ferric ions (HTA). We found that HTA had better photothermal efficiency than a pure hydrogel; its photothermal repeatability is good and it can be NIR (808 nm) irradiated as needed. In addition, the precooled hydrogel solution can be injected into the tumor and it can rapidly gel in situ. In vitro, HTA with NIR irradiation (HTA + NIR) induced the apoptosis of K7M2 cancer cells. In vivo, the local administration of HTA + NIR exerted photothermal killing of primary tumors and reprogramming of TAMs into M1-type macrophages in the TME. Therefore, the injectable photothermally active antitumor hydrogel has great potential for modulating the TME to treat bone tumors.

Injectable immunomodulation-based porous chitosan microspheres/HPCH hydrogel composites as a controlled drug delivery system for osteochondral regeneration.

The inappropriate regenerated fibrous cartilage and subchondral bone of the injured chondral defect ultimately cause degeneration of the regenerated cartilage, which eventually leads to the failure of cartilage repair. In this study, we developed a macrophage-modulated and injectable 'building block' drug delivery system comprised of porous chitosan (CS) microspheres and hydroxypropyl chitin (HPCH) hydrogel, where the dimethyloxallyl glycine (DMOG) was encapsulated in the thermosensitive HPCH hydrogel (HD) while kartogenin (KGN) was conjugated on the porous CS microspheres (CSK-PMS). The developed HD/CSK-PMS composite scaffold effectively modulated the microenvironment at the defect site, achieved local macrophage M2 polarization and promoted cartilage regeneration. The fast-degradable HD favored hyaline cartilage regeneration, while the highly stable CSK-PMS supported the endochondral ossification and regenerated the subchondral bone. In vitro and in vivo evaluations revealed that the newly developed HD/CSK-PMS as a controlled drug delivery system could effectively create M2 macrophage microenvironment and orchestrate osteochondral (OC) regeneration. These findings indicate the importance of the immune microenvironment and subchondral bone for high-quality cartilage repair, and thus the immunomodulation-based hydrogel/PMS composite system could be a promising candidate for OC regeneration.

Hierarchical-structured bacterial cellulose/potato starch tubes as potential small-diameter vascular grafts.

To achieve long-term patent small-diameter (<6 mm) vascular implants, biomimetic vascular grafts have gained much attention in promoting in situ blood vessel regeneration. In this study, hierarchical-structured bacterial cellulose/potato starch (BC/PS) composites were biosynthesized by the addition of swollen PS. Investigations on the physicochemical properties of BC/PS composites showed that the properties could be improved and tailored by the addition of swollen PS. The composites displayed a morphology, water content, thermal properties, mechanical properties, and biocompatibility appropriate for vascular tissue engineering. Most importantly, the BC/PS grafts, with a dense inner surface and a circumferential macroporous outer layer, possessed 75% patency and promoted rapid blood vessel regeneration in in vivo assessment on rabbits, with complete endothelium monolayer, organized smooth muscle cells, rich new capillaries, and deposited extracellular matrix. Collectively, these findings demonstrate that hierarchical-structured BC/PS tubes hold great promise as artificial small-diameter vascular grafts.

Biomimetic Ti-6Al-4V alloy/gelatin methacrylate hybrid scaffold with enhanced osteogenic and angiogenic capabilities for large bone defect restoration.

Titanium-based scaffolds are widely used implant materials for bone defect treatment. However, the unmatched biomechanics and poor bioactivities of conventional titanium-based implants usually lead to insufficient bone integration. To tackle these challenges, it is critical to develop novel titanium-based scaffolds that meet the bioadaptive requirements for load-bearing critical bone defects. Herein, inspired by the microstructure and mechanical properties of natural bone tissue, we developed a Ti-6Al-4V alloy (TC4)/gelatin methacrylate (GelMA) hybrid scaffold with dual bionic features (GMPT) for bone defect repair. GMPT is composed of a hard 3D-printed porous TC4 metal scaffold (PT) backbone, which mimics the microstructure and mechanical properties of natural cancellous bone, and a soft GelMA hydrogel matrix infiltrated into the pores of PT that mimics the microenvironment of the extracellular matrix. Ascribed to the unique dual bionic design, the resultant GMPT demonstrates better osteogenic and angiogenic capabilities than PT, as confirmed by the in vitro and rabbit radius bone defect experimental results. Moreover, controlling the concentration of GelMA (10%) in GMPT can further improve the osteogenesis and angiogenesis of GMPT. The fundamental mechanisms were revealed by RNA-Seq analysis, which showed that the concentration of GelMA significantly influenced the expression of osteogenesis- and angiogenesis-related genes via the Pi3K/Akt/mTOR pathway. The results of this work indicate that our dual bionic implant design represents a promising strategy for the restoration of large bone defects.

Preparation and evaluation of ion-exchange porous polyvinyl alcohol microspheres as a potential drug delivery embolization system.

The present study aimed to develop a new drug delivery system with efficient drug loading and sustained drug release for potential application in transarterial chemoembolization (TACE). The porous polyvinyl alcohol microspheres (PPVA MS) were prepared by a combination of inverse emulsification and thermal-induced phase separation (TIPS) method, this was followed by the grafting polymerization of sodium 4-styrene sulfonate (SSS) onto the PPVA MS to obtain the grafted PPVA-g-PSSS MS. The prepared PPVA MS showed a well-defined spherical shape with 'honeycomb-like' porous structure, which could be readily tailored by adjusting the quenching temperature. In vitro biocompatibility analysis indicated the non-cytotoxic and hemocompatible nature of PPVA MS. The porous structure and presence of ionically charged groups in the PPVA-g-PSSS MS favoured the loading of cationic doxorubicin (DOX) onto the MS through ionic-interactions and demonstrated a sustained drug release pattern. Moreover, the cytotoxicity of DOX-loaded PPVA-g-PSSS (DOX@PPVA-g-PSSS) MS against HepG2 cells and the intracellular uptake of DOX demonstrated the potent in vitro antitumor activity. Furthermore, the central auricular artery embolization in rabbits showed that both the PPVA-g-PSSS and DOX@PPVA-g-PSSS MS could occlude the auricular arteries and induced superior embolization effects, such as progressive ear appearance changes, irreversible parenchymal damage and fibrosis, and ultrastructural alternations in endothelial cells. Besides, the DOX fluorescence was distributed around the embolized arteries, without decreasing its intensity when prolonged embolization up to 15 days. These findings suggest that the newly developed DOX@PPVA-g-PSSS MS could be employed as a promising drug-loaded embolic agent for the treatment of hepatocellular carcinoma.

A pH-response chemotherapy synergistic photothermal therapy for tumor suppression and bone regeneration by mussel-inspired Mg implant.

Primary malignant bone tumors can be life-threatening. Surgical resection of tumor plus chemotherapy is the standard clinical treatment. However, postoperative recovery is hindered due to tumor recurrence caused by residual tumor cells and bone defect caused by resection of tumor tissue. Herein, a multifunctional mussel-inspired film was fabricated on Mg alloy, that is, an inner hydrothermal-treated layer, a middle layer of polydopamine, and an outer layer of doxorubicin. The modified Mg alloy showed excellent photothermal effect and thermal/pH-controlled release of doxorubicin. The synergistic effect of chemotherapy and photothermal therapy enabled the modified Mg alloy to kill bone tumor in vitro and inhibit tumor growth in nude mice. Moreover, because of the controlled release of Mg ions and biocompatibility of polydopamine, the modified Mg alloy supported extracellular matrix mineralization, alkaline phosphatase activity, and bone-related gene expression in C3H10T1/2. Bone implantation model in rats verified that the modified Mg showed excellent osteointegration. These findings prove that the use of mussel-inspired multifunction film on Mg alloy offers a promising strategy for the therapy of primary malignant bone tumor.

Immobilized thrombin on X-ray radiopaque polyvinyl alcohol/chitosan embolic microspheres for precise localization and topical blood coagulation.

Trans-catheter arterial embolization (TAE) plays an important role in treating various diseases. The available embolic agents lack X-ray visibility and do not prevent the reflux phenomenon, thus hindering their application for TAE therapy. Herein, we aim to develop a multifunctional embolic agent that combines the X-ray radiopacity with local procoagulant activity. The barium sulfate nanoparticles (BaSO4 NPs) were synthesized and loaded into the polyvinyl alcohol/chitosan (PVA/CS) to prepare the radiopaque BaSO4/PVA/CS microspheres (MS). Thereafter, thrombin was immobilized onto the BaSO4/PVA/CS MS to obtain the thrombin@BaSO4/PVA/CS MS. The prepared BaSO4/PVA/CS MS were highly spherical with diameters ranging from 100 to 300 µm. In vitro CT imaging showed increased X-ray visibility of BaSO4/PVA/CS MS with the increased content of BaSO4 NPs in the PVA/CS MS. The biocompatibility assessments demonstrated that the MS were non-cytotoxic and possessed permissible hemolysis rate. The biofunctionalized thrombin@BaSO4/PVA/CS MS showed improved hemostatic capacity and facilitated hemostasis in vitro. Additionally, in vivo study performed on a rabbit ear embolization model confirmed the excellent X-ray radiopaque stability of the BaSO4/PVA/CS MS. Moreover, both the BaSO4/PVA/CS and thrombin@BaSO4/PVA/CS MS achieved superior embolization effects with progressive ischemic necrosis on the ear tissue and induced prominent ultrastructural changes in the endothelial cells. The findings of this study suggest that the developed MS could act as a radiopaque and hemostatic embolic agent to improve the embolization efficiency.

Immobilizing magnesium ions on 3D printed porous tantalum scaffolds with polydopamine for improved vascularization and osteogenesis.

Large bone defects remain a worldwide healthy problem needing to be solved. 3D printed tantalum (Ta) scaffolds have enormous potential to repair bone defects and have applied in clinic in recent years. Although the porous structure of 3D printed Ta scaffolds could allow bone ingrowth, the surface property that reactive with surrounding tissue is still unfavorable and thus the early osteointegration is impeded. Magnesium (Mg), a necessary element during bone development, has been reported with effectively osteogenesis and angiogenesis capacity. Hence, in this study, three concentrations of Mg were doped on the surface of 3D printed tantalum scaffolds utilizing the surface adhesion ability of polydopamine (Ta-PDA-Mg) to improve its surface bioactivity. The physiochemical property of resultant Ta-PDA-Mg scaffold was characterized and their osteogenic and angiogenic effects were tested through a serial of experiments both in vitro and in vivo. Results show that Ta-PDA-Mg2 possessed the highest ion release, and all scaffolds showed excellent biocompatibility. The adhesion, angiogenesis and osteogenesis were all improved in Mg doping groups in vitro, while the Ta-PDA-Mg2 exhibited the best performances. Then the in vivo performance was examined through rat femur condyles bone defect model. Results demonstrate that the Ta-PDA-Mg2 significantly enhanced the vascularized bone formation and the osteointegration, which was further confirmed through pull out test. Therefore, Mg doped 3D printed Ta scaffold could improve surface bioactivity and lead to better osteogenesis and angiogenesis, which may provide novel strategy to develop bioactive customized implants in orthopedic applications.

Development of finasteride/PHBV@polyvinyl alcohol/chitosan reservoir-type microspheres as a potential embolic agent: from in vitro evaluation to animal study.

Benign prostatic hyperplasia (BPH) is a prevalent urological disease affecting elders. Currently, the prostatic artery embolization (PAE) is considered as a minimally invasive and safe technique to treat BPH. However, various drug-loaded embolic agents have not been thoroughly investigated in BPH therapy. In this study, finasteride/poly(3-hydroxybutyrate-3-hydroxyvalerate)@polyvinyl alcohol/chitosan (FNS/PHBV@PVA/CS) reservoir-type microspheres were prepared via the solid-in-water-in-oil (S/W/O) emulsion crosslinking method with the aim to reduce the burst effect and control localized drug delivery. The structure and properties of the drug and resultant microspheres were characterized via field emission scanning electron microscopy (FESEM), Fourier-transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), and thermogravimetric analysis (TGA). The results showed that the drug-loaded hybrid microspheres were well-dispersed and spherical with a mean diameter of 238.1 ± 27.3 µm. All samples exhibited excellent thermal stability. The FNS/PHBV microspheres were successfully encapsulated inside the PVA/CS polymeric matrix, which effectively suppressed the burst effect and prolonged the drug release up to 51 days. In vitro biocompatibility assessment indicated that the microspheres possessed excellent cytocompatibility and hemocompatibility. Furthermore, in vivo studies performed in the rabbit ear embolization model showed the formation of progressive ischemic necrosis after treatment for various periods. Histopathological studies revealed that the microspheres completely occluded the blood vessels with minimal foreign body response and formed the fibrotic area at the periphery of embolized arteries. Furthermore, the auricular vascular endothelial cells showed acute ultrastructural changes, associated with the ischemic necrosis induced by the embolization procedures. All these findings suggest that the FNS/PHBV@PVA/CS hybrid microspheres could be used as a promising drug delivery system for potential applications in BPH therapy.

Cartilage repair mediated by thermosensitive photocrosslinkable TGFß1-loaded GM-HPCH via immunomodulating macrophages, recruiting MSCs and promoting chondrogenesis.

Repairing cartilage defects using thermosensitive hydrogels is an attractive treatment strategy, but the poor mechanical properties and limited understanding of the interactions between hydrogels and cells limit their application. Methods: In this study, a thermosensitive hydroxypropyl chitin hydrogel (HPCH) was functionalized with methacrylate groups to synthesize photocrosslinkable glycidyl methacrylate-modified HPCH (GM-HPCH). GM-HPCH could form a gel in situ through a thermosensitive sol-gel transition and its mechanical properties can be improved by UV irradiation. Cell viability, cell adhesion and anti-apoptosis activity of GM-HPCH were evaluated. Transforming growth factor-ß1 (TGFß1) was introduced into the GM-HPCH hydrogel to fabricate the composite hydrogel. The macrophage immunomodulation, MSC recruitment and chondrogenesis of the composite hydrogel were evaluated. Results: With high biocompatibility, GM-HPCH could protect chondrocytes from apoptosis. Both the in vitro and in vivo experiments showed that GM-HPCH + TGFß1 shifted the recruited macrophages from M1 to M2 and promoted chondrogenic gene expression. Additionally, the composite hydrogel could promote the migration of marrow stromal cells (MSCs) in the Transwell test and increase migrated gene expression. The fluorescent tracking of MSCs confirmed MSC homing in the rat chondral defect with the help of GM-HPCH. The macroscopic evaluation and histological results at 6 weeks and 12 weeks postsurgery showed that GM-HPCH + TGFß1 can achieve superior cartilage healing. Conclusions: The GM-HPCH + TGFß1 hydrogel effectively promoted cartilage repair via immunomodulating macrophages, recruiting MSCs and promoting chondrogenesis; thus it is a promising injectable hydrogel for cartilage regeneration.

Mesenchymal stem cell-loaded thermosensitive hydroxypropyl chitin hydrogel combined with a three-dimensional-printed poly(ε-caprolactone) /nano-hydroxyapatite scaffold to repair bone defects via osteogenesis, angiogenesis and immunomodulation.

Chitin-derived hydrogels are commonly used in bone regeneration because of their high cell compatibility; however, their poor mechanical properties and little knowledge of the interaction between the materials and host cells have limited their practical application. Methods: To evaluate osteoinductivity and enhance the mechanical properties of a newly synthesized thermosensitive hydroxypropyl chitin hydrogel (HPCH), a mesenchymal stem cell (MSC)-encapsulated HPCH was infused into a three-dimensional-printed poly (ε-caprolactone) (PCL)/ nano-hydroxyapatite (nHA) scaffold to form a hybrid scaffold. The mechanical properties and cell compatibility of the scaffold were tested. The interaction between macrophages and scaffold for angiogenesis and osteogenesis were explored in vitro and in vivo. Results: The hybrid scaffold showed improved mechanical properties and high cell viability. When MSCs were encapsulated in HPCH, osteo-differentiation was promoted properly via endochondral ossification. The co-culture experiments showed that the hybrid scaffold facilitated growth factor secretion from macrophages, thus promoting vascularization and osteoinduction. The Transwell culture proved that MSCs modulated the inflammatory response of HPCH. Additionally, subcutaneous implantation of MSC-encapsulated HPCH confirmed M2 activation. In situ evaluation of calvarial defects confirmed that the repair was optimal in the MSC-loaded HPCH + PCL/nHA group. Conclusions: PCL/nHA + HPCH hybrid scaffolds effectively promoted vascularization and osteoinduction via osteogenesis promotion and immunomodulation, which suggests promising applications for bone regeneration.

Tantalum nanoparticles reinforced polyetheretherketone shows enhanced bone formation.

Polyetheretherketone (PEEK) has been used in orthopedic surgery for several decades. Numerous methods were invented to alter the properties of PEEK. By adding nanoparticles, fibers, etc., elastic modulus and strength of PEEK can be changed to meet certain demand. In this study, tantalum (Ta), a promising metal, was introduced to modify the properties of PEEK, in which PEEK was reinforced with different contents of tantalum nanoparticles (from 1 wt% to 9 wt%). Mechanical properties and biological functions (both in vitro and in vivo) were then investigated. The highest elastic modulus and compressive strength were observed in 3%Ta-PEEK. Cell experiments as cell adhesion, collagen secretion, biomineralization and osteogenesis related gene expression showed preferable results in 3%Ta-PEEK and 5%Ta-PEEK. Improved bone integration was shown in 3%Ta-PEEK and 5%Ta-PEEK in vivo. Above all, enhanced mechanical properties and promoted bone formation were proved for 3%Ta-PEEK and 5%Ta-PEEK compared to others groups both in vitro and in vivo, suggesting that the addition of tantalum nanoparticles modified the osseointegration ability of PEEK. This composite of tantalum and PEEK could have a clinical potential for orthopedic implants.