Construction of millimeter-scale vascularized engineered myocardial tissue using a mixed gel.

Engineering myocardium has shown great clinal potential for repairing permanent myocardial injury. However, the lack of perfusing blood vessels and difficulties in preparing a thick-engineered myocardium result in its limited clinical use. We prepared a mixed gel containing fibrin (5 mg/ml) and collagen I (0.2 mg/ml) and verified that human umbilical vein endothelial cells (HUVECs) and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could form microvascular lumens and myocardial cell clusters by harnessing the low-hardness and hyperelastic characteristics of fibrin. hiPSC-CMs and HUVECs in the mixed gel formed self-organized cell clusters, which were then cultured in different media using a three-phase approach. The successfully constructed vascularized engineered myocardial tissue had a spherical structure and final diameter of 1-2 mm. The tissue exhibited autonomous beats that occurred at a frequency similar to a normal human heart rate. The internal microvascular lumen could be maintained for 6 weeks and showed good results during preliminary surface re-vascularization in vitro and vascular remodeling in vivo. In summary, we propose a simple method for constructing vascularized engineered myocardial tissue, through phased cultivation that does not rely on high-end manufacturing equipment and cutting-edge preparation techniques. The constructed tissue has potential value for clinical use after preliminary evaluation.

Preclinical evaluation of the safety and effectiveness of a new bioartificial cornea.

Cross-linking agents are frequently used to restore corneal properties after decellularization, and it is especially important to select an appropriate method to avoid excessive cross-linking. In addition, how to promote wound healing and how to improve scar formation require further investigation. To ensure the safety and efficacy of animal-derived products, we designed bioartificial corneas (BACs) according to the criteria for Class III medical devices. Our BACs do not require cross-linking agents and increase mechanical strength via self-cross-linking of aldehyde-modified hyaluronic acid (AHA) and carboxymethyl chitosan (CMC) on the surface of decellularized porcine corneas (DPCs). The results showed that the BACs had good biocompatibility and transparency, and the modification enhanced their antibacterial and anti-inflammatory properties in vitro. Preclinical animal studies showed that the BACs can rapidly regenerate the epithelium and restore vision within a month. After 3 months, the BACs were gradually filled with epithelial, stromal, and neuronal cells, and after 6 months, their transparency and histology were almost normal. In addition, side effects such as corneal neovascularization, conjunctival hyperemia, and ciliary body hyperemia rarely occur in vivo. Therefore, these BACs show promise for clinical application for the treatment of infectious corneal ulcers and as a temporary covering for corneal perforations to achieve the more time.

A dual-functional implant with an enzyme-responsive effect for bacterial infection therapy and tissue regeneration.

Biomaterial-associated bacterial infection is one of the major causes of implant failure. The treatment of such an implant infection typically requires the elimination of bacteria and acceleration of tissue regeneration around implants simultaneously. To address this issue, an ideal implanted material should have the dual functions of bacterial infection therapy and tissue regeneration at the same time. Herein, an enzyme-responsive nanoplatform was fabricated in order to treat implant-associated bacterial infection and accelerate tissue regeneration in vivo. Firstly, Ag nanoparticles were pre-encapsulated in mesoporous silica nanoparticles (MSNs) by a one-pot method. Then, poly-l-glutamic acid (PG) and polyallylamine hydrochloride (PAH) were assembled by the layer-by-layer (LBL) assembly technique on MSN-Ag to form LBL@MSN-Ag nanoparticles. Furthermore, the LBL@MSN-Ag nanoparticles were deposited on the surface of polydopamine-modified Ti substrates. PG is a homogeneous polyamide composed of an amide linkage, which can be degraded by glutamyl endonuclease secreted by Staphylococcus aureus. Inductively coupled plasma spectroscopy (ICP) results proved that the LBL@MSN-Ag particles show a significant enzyme responsive release of Ag ions. Furthermore, results of antibacterial experiments in vitro showed that the Ti substrates modified with an LBL@MSN-Ag nanocoating presented an excellent antibacterial effect. As for an animal experiment in vivo, in a bacterium infected femur-defect rat model, the modified Ti implants effectively treated bacterial infection. More importantly, the results of micro-CT, haematoxylin-eosin staining and Masson's trichrome staining demonstrated that the modified Ti implants significantly promoted the formation of new bone tissue after implantation for 4 weeks. The present system paves the way for developing the next generation of implants with the functions of treating bacterial infection and promoting tissue regeneration.

Fabrication of enzyme-responsive composite coating for the design of antibacterial surface.

In this study, a type of bacteria enzyme-triggered antibacterial surface with a controlled release of Ag ions was developed. Firstly, chitosan-silver nanocomposites (Chi@Ag NPs) were in situ synthesized via using ascorbic acid as reducing agent. Chi@Ag NPs were characterized by transmission electron microscopy, ultraviolet-visible spectroscopy, X-ray diffraction and X-ray photoelectron spectroscopy. Subsequently, Chi@Ag NPs and hyaluronic acid (HA) were used to fabricate antibacterial composite coating via Layer-by-Layer (LBL) self-assembly method. The successful construction of Chi@Ag NPs/HA composite coating was confirmed by scanning electron microscopy, energy dispersive spectroscopy and contact angle measurements, respectively. Then, the amount of released Ag ion was analyzed by inductively coupled plasma atomic emission spectrometry, which demonstrated that the release of Ag ions from the surface could be triggered by enzyme (e.g. hyaluronidase). A series of antibacterial tests in vitro, including zone of inhibition test, bacterial viability assay, antibacterial rate measurement and bacteria adhesion observation, demonstrated that the enzyme-responsive surface could inhibit the growth of bacteria. On the whole, this study provides an alternative approach for the fabrication of antibacterial surfaces on synthetic materials in various fields with the minimal side effects on surrounding environment and human body.

Construction of Ag-incorporated coating on Ti substrates for inhibited bacterial growth and enhanced osteoblast response.

In orthopedic fields, effective anti-infection property and promotive biocompatibility on surface of titanium implants are two crucial factors for long-term successful implants. Herein, Ag nanoparticles (NPs) loaded TiO2 nanotubes (TNT) arrays were fabricated on Ti substrates with assistance of ultraviolet irradiation. Then, bioactive multilayer films of chitosan (CHI) and dialdehyde alginate (ADA) pair were deposited onto the Ag-loaded TNT arrays via a layer-by-layer (LBL) self-assembly technique, which could effectively achieve the impactful antibacterial ability of titanium and endow the substrates with favorable biocompatibility. The driving force of the assembling of multilayer films came from two sources, electrostatic interaction and covalent interaction of Schiff-bonds between CHI and ADA. The surface topography and wettability of different samples were characterized by field emission scanning electron microscopy, transmission electron microscopy and contact angle measurements, respectively. In addition, Ag ions release from TNT-Ag and LBL substrate was measured via inductively coupled plasma atomic emission spectroscopy (ICP-AES). The results of a series of biological behaviors of osteoblasts on different substrates in vitro, including lactate dehydrogenase activity assay, cytoskeleton observation and cell viability measurement, confirmed that LBL substrates coated with (ADA-CHI)10 multilayer films have negligible cytotoxicity and promote osteoblast growth compared with TNT-Ag substrates, which could ascribe to the slow-release of Ag ions and the biocompatibility of (ADA-CHI)10 multilayer. More importantly, owing to the release of Ag ions, the LBL samples still exhibited a prominent antibacterial activity for S.aureus and E.coli. Characteristics of bacterial adhesion and viability measurement proved that the fabricated Ag-incorporated platform was capable of obviously inhibiting the adhesion and growth of bacteria. Therefore, this approach of surface modification for Ti substrates presented here may provide an alternative strategy to simultaneously meet the desirable osteoblast growth and reduce bacterial infection for implants in clinical application.

Investigation of osteogenic responses of Fe-incorporated micro/nano-hierarchical structures on titanium surfaces.

Effective and fast osseointegration is important for the survival of titanium-based orthopaedic implants. Previous studies confirmed that topographic features combined with inorganic ions showed positive effects on the biological functions of osteoblastic cells. In this study, we report an approach for fabricating Fe-incorporated micro-nano hierarchical structures on titanium substrates, which was realized by dual acid etching and subsequent hydrothermal treatment. The surface morphology, surface chemistry and wettability of the titanium substrates were characterized using scanning electron microscopy, X-ray photoelectron spectroscopy and contact angle measurements, respectively. The Fe-incorporated micro-nano hierarchical titanium substrates were probed to be biocompatible and positively improved protein adsorption, cell proliferation and cell differentiation of osteoblasts in vitro. Furthermore, the Fe-incorporated titanium substrates significantly enhanced the expressions of osteogenic genes (such as Runx2, Col I, OPN, and OCN), which were attributed to the synergistic effects of micro-nano structures and Fe ions. More importantly, the Fe-incorporated titanium implants with micro-nano hierarchical structures promoted new bone formation in vivo. This study provides an alternative for the development of orthopaedic implants with improved osseointegration for potential clinical applications.

Surface modification of titanium substrates for enhanced osteogenetic and antibacterial properties.

The insufficient osseointegration and bacterial infection of titanium and its alloys remain the key challenges in their clinic applications, which may result in failure implantation. To improve osteogenetic and antibacterial properties, TiO2 nanotube arrays were fabricated on titanium substrates for loading of antibacterial drug. Then, TiO2 nanotube arrays were covered with chitosan/sodium alginate multilayer films. The successful construction of this system was verified via scanning electron microscopy and contact angle measurement. The cytocompatibility evaluation in vitro, including cytoskeleton observation, cell viability measurement, and alkaline phosphatase activity assay, confirmed that the present system was capable of accelerating the growth of osteoblasts. In addition, bacterial adhesion and viability assay verified that treated Ti substrates were capable of reducing the adhesion of bacteria. This study may provide an alternative to develop titanium-based implants for enhanced bone osseointegration and reduced bacterial infection.