ACS Biomaterials Science & Engineering最新文献

In recent years, there has been increased interest in the use of cellular spheroids, microtissues, and organoids as biological building blocks to engineer functional tissues and organs. Such microtissues are typically formed by the self-assembly of cellular aggregates and the subsequent deposition of a tissue-specific extracellular matrix (ECM). Biofabrication and 3D bioprinting strategies using microtissues may require the development of supporting hydrogels and bioinks to spatially localize such biological building blocks in 3D space and hence enable the engineering of geometrically defined tissues. Therefore, the aim of this work was to engineer scaled-up, geometrically defined cartilage grafts by combining multiple cartilage microtissues within a rapidly degrading oxidized alginate (OA) supporting hydrogel and maintaining these constructs in dynamic culture conditions. To this end, cartilage microtissues were first independently matured for either 2 or 4 days and then combined in the presence or absence of a supporting OA hydrogel. Over 6 weeks in static culture, constructs engineered using microtissues that were matured independently for 2 days generated higher amounts of glycosaminoglycans (GAGs) compared to those matured for 4 days. Histological analysis revealed intense staining for GAGs and negative staining for calcium deposits in constructs generated by using the supporting OA hydrogel. Less physical contraction was also observed in constructs generated in the presence of the supporting gel; however, the remnants of individual microtissues were more observable, suggesting that even the presence of a rapidly degrading hydrogel may delay the fusion and/or the remodeling of the individual microtissues. Dynamic culture conditions were found to modulate ECM synthesis following the OA hydrogel encapsulation. We also assessed the feasibility of 3D bioprinting of cartilage microtissues within OA based bioinks. It was observed that the microtissues remained viable after extrusion-based bioprinting and were able to fuse after 48 h, particularly when high microtissue densities were used, ultimately generating a cartilage tissue that was rich in GAGs and negative for calcium deposits. Therefore, this work supports the use of OA as a supporting hydrogel/bioink when using microtissues as biological building blocks in diverse biofabrication and 3D bioprinting platforms.

Embedded 3D printing (EMB3D) is an additive manufacturing technique that enables complex fabrication of soft materials including tissues and silicones. In EMB3D, a nozzle writes continuous filaments into a support bath consisting of a yield stress fluid. Lack of fusion defects between filaments can occur because the nozzle pushes support fluid into existing filaments, preventing coalescence. Interfacial tension was previously proposed as a tool to drive interfilament fusion. However, interfacial tension can also drive rupture and shrinkage of printed filaments. Here, we evaluate the efficacy of interfacial tension as a tool to control defects in EMB3D. Using polydimethylsiloxane (PDMS)-based inks with varying amounts of fumed silica and surfactant, printed into Laponite in water supports, we evaluate the effect of rheology, interfacial tension, print speeds, and interfilament spacings on defects. We print pairs of parallel filaments at varying orientations in the bath and use digital image analysis to quantify shrinkage, rupture, fusion, and positioning defects. By comparing disturbed filaments to printed pairs of filaments, we disentangle the effects of nozzle movement and filament extrusion. Critically, we find that capillary instabilities and interfilament fusion scale with the balance between support rheology and interfacial tension. Less viscous supports and higher interfacial tensions lead to more shrinkage and rupture at all points in the printing process, from relaxation after writing, to disturbance of the line, to writing of a second line. It is necessary to overextrude material to achieve interfilament fusion, particularly at high support viscosities and low interfacial tensions. Finally, fusion quality varies with printing orientation, and writing neighboring filaments causes displacement of existing structures. As such, specialized slicers are needed for EMB3D that consider the tighter spacings and orientation-dependent spacings necessary to achieve precise control over printed shapes.

Rapid real-time PCR (generally <1 h) has broad prospects. In this study, we synthesized a new type of nanomaterial core-shell tecto-dendrimer coated with Au nanoparticles (Au CSTDs) for research in this field. The experimental results showed that Au CSTDs could significantly shorten the time of real-time PCR (from 72 to 28 min) with different templates, while the detection limit reached 10 copies and the nonspecific amplification was significantly reduced. Furthermore, experimental analyses and theoretical studies using the finite element simulation method confirmed that Au CSTDs function by synergistically enhancing electrostatic adsorption and thermal conductivity. These properties play a key role in improving real-time PCR, especially in particle-particle interactions. This study contributes an advanced method to rapid real-time PCR, which is expected to remarkably improve the efficiency, lower the detection limit, and enhance the specificity of molecular detection.

Droplets, tiny liquid compartments, are increasingly emerging in the biomedical and biomanufacturing fields due to their unique properties to serve as templates or independent reaction units. Currently, the straightforward and efficient generation of various functional droplets in a biofriendly manner remains challenging. Herein, a novel microfluidic-assisted pneumatic strategy is described for the customizable and high-throughput production of monodispersed droplets, and the droplet size can be precisely controlled via a simplified gas pressure regulation module. In particular, numerous uniform alginate microcarriers can be rapidly fabricated in an all-aqueous manner, wherein the encapsulated islet or liver cells exhibit favorable viability and biological functions. Furthermore, by changing the microchannel configuration, several fluid manipulation functions developed by microfluidic technology, such as mixing and laminar flow, can be successfully incorporated into this platform. The droplet generators with scalable functionality are demonstrated in many biomanufacturing scenarios, including on-demand distribution of cell-mimetic particles, continuous synthesis of biomedical metal-organic framework (MOF), controllable preparation of compartmental microgel, etc. These may provide sustainable inspiration for developing droplet generators and their applications in tissue and organ engineering, biomaterials design, bioprinting nozzles, and other fields.

Mitochondria are essential for cellular functions, such as energy production. Human mitochondrial DNA (mtDNA), encoding 13 distinct genes, two rRNA, and 22 tRNA, is crucial for maintaining vital functions, along with nuclear-encoded mitochondrial proteins. However, mtDNA is prone to somatic mutations due to replication errors and reactive oxygen species exposure. These mutations can accumulate, leading to heteroplasmic conditions associated with severe metabolic diseases. Therefore, developing methodologies to improve mitochondrial health is highly demanded. Introducing nucleic acids directly into mitochondria is a promising strategy to control mitochondrial gene expression. Messenger RNA (mRNA) delivery especially offers several advantages such as faster gene expression and reduced risk of genome integration if accidentally delivered to the cell nucleus. In this study, we investigated the effect of the poly(A) tail length of mRNA on the mitochondrial translation to achieve efficient expression. We used a peptide-based mitochondrial targeting system, mitoNEET-(RH)₉, comprising a mitochondria-targeting sequence (MTS) and a cationic sequence, to deliver mRNA with various poly(A) tails into the mitochondria. The poly(A) tail length significantly affected translational efficiency, with a medium length of 60 nucleotides maximizing protein expression in various cell lines due to enhanced interaction with mitochondrial RNA-binding proteins. Our findings highlight the importance of optimizing poly(A) tail length for efficient mitochondrial mRNA translation, providing a potential strategy for improving mitochondrial gene therapy. These results pave the way for further exploration of the mechanisms and clinical applications of mitochondrial mRNA delivery systems.

Subcutaneous implants can provide patients with long-acting, compliance-independent drug dosing. For this reason, subcutaneous implants have shown emerging interest in human immunodeficiency virus (HIV) prevention. However, any successful long-acting HIV-prevention device will require multidrug dosing, which poses a challenge for formulation considering the physicochemically diverse selection of antiretroviral (ARV) candidates. As a method that has shown the capacity of efficient multidrug delivery, we assessed electrospun fiber implants composed of three synergistically potent ARVs and a biodegradable polymer selected by in vitro release studies. In mice, subcutaneous electrospun fiber implants exhibit burst release of the more hydrophilic drugs maraviroc (MVC) and raltegravir (RAL), which could be reduced via simple prewash treatments of the implants. Over an extended 120 day time frame, fiber implants show drug-specific differences in release time frames and magnitudes in blood serum. However, end-point drug tissue concentrations show that the most hydrophobic drug etravirine (ETR) remains in high concentrations within the implant and in local skin tissue biopsies. Furthermore, ETR is found to be capable of significant partitioning into lymph nodes, the lower female reproductive tract, and the rectum. Topologically smooth film implants also exhibit the same drug-dependent trends. Therefore, we illustrate that drug release and drug tissue partitioning are largely dictated by drug properties. Further, we find that the properties of ETR enable significant drug quantities within the tissues most relevant to HIV protection. Evidence from this work emphasizes the need for a greater focus on drug properties and prodrug strategies to enable relevant, extended, and targeted drug release.

Scaffolds play a crucial role in bone tissue engineering to support the defect area through bone regeneration and defect reconstruction. Promising tissue regeneration without negative repercussions and avoidance of the lifelong presence inside the body make bioresorbable metals prosper in the field of regenerative medicine. Recently, Zn and its alloys have emerged as promising biodegradable materials for their moderate degradation rate and satisfactory biocompatibility. Nevertheless, it is very challenging for cells to adhere and grow over the Zn surface alone, which influences the tissue-implant integration. In this study, an attempt has been made to systematically investigate the bioactivity responses in terms of in vitro hemocompatibility, cytotoxicity, antibacterial activity, and in vivo biocompatibility of newly developed Zn-2Cu-0.5Mn/Mg alloy scaffolds with different surface roughness. The rough surface of Zn-2Cu-0.5Mg shows the highest degradation rate of 0.16 mm/yr. The rough surface exhibits a prominent role in the adsorption of protein, further enhancing cell adhesion. Concentration-dependent alloy extract shows the highest cell proliferation for 12.5% of the extract with a maximum cell viability of 101% in Zn-2Cu-0.5Mn and 108% in Zn-2Cu-0.5Mg after 3 d. Acceptable hemolysis percentages (less than 5%) with promising anticoagulation properties are observed for all of the conditions. Enhanced antibacterial (Staphylococcus aureus and Escherichia coli) activity due to a significant effect of ions illustrates the maximum killing effect on the bacterial colony for the rough Zn-2Cu-0.5Mg alloy. In addition, it is observed that for rough Zn-2Cu-0.5Mn/Mg alloys, the inflammatory response is minimal after subcutaneous implantation, and neo-bone tissue forms in the defect areas of the rat femur with satisfactory biosafety response. The osseointegration property of the Zn-2Cu-0.5Mg alloy is comparable to that of the Zn-2Cu-0.5Mn alloy. Therefore, the rough surface of the Zn-2Cu-0.5Mg alloy has the potential to enhance biocompatibility and promote better osseointegration activity with host tissues for various biomedical applications.

The integration of hydrogel-based bioinks with 3D bioprinting technologies presents an innovative approach to chronic wound management, which is particularly challenging to treat because of its multifactorial nature and high risk of complications. Using precise deposition techniques, 3D bioprinting significantly alters traditional wound care paradigms by enabling the fabrication of patient-specific wound dressings that imitate natural tissue properties. Hydrogels are notably beneficial for these applications because of their abundant water content and mechanical properties, which promote cell viability and pathophysiological processes of wound healing, such as re-epithelialization and angiogenesis. This article reviews key 3D printing technologies and their significance in enhancing the structural and functional outcomes of wound-care solutions. Challenges in bioink viscosity, cell viability, and printability are addressed, along with discussions on the cross-linking and mechanical stability of the constructs. The potential of 3D bioprinting to revolutionize chronic wound management rests on its capacity to generate remedies that expedite healing and minimize infection risks. Nevertheless, further studies and clinical trials are necessary to advance these therapies from laboratory to clinical use.

We successfully synthesized folic acid (FA) immobilized hydroxyapatite (HA) nanoparticles without using a mediative reagent (e.g., silane coupling agent), and the immobilization states were evaluated and discussed. The HA nanoparticles with higher biocompatibility have two different planes, namely, c- and m-planes. These plane surfaces are rich in phosphate groups (P-site) and Ca²⁺ ions (C-site), respectively. We suggested that during the synthesis of the HA nanoparticles, the P-site substitution and C-site coordination with the addition of organic molecules containing -COO^- ions can occur. Thus, it is possible to simultaneously immobilize two molecules to one HA nanoparticle. In this study, we successfully synthesized FA-immobilized HA nanoparticles by P-site substitution and C-site coordination reactions, which were named as substitution type and coordination type. In the substitution type, when FA was reacted with HA during the nucleation stage, the PO₄^3- ions of HA decreased as the FA ratio of coverage surface area increased, and the crystalline phase was changed significantly from the Ca deficient HA to the carbonated HA phase. Accordingly, it was indicated that FA was immobilized on HA by the P-site substitution. In the coordination type, since FA was reacted with HA after the completion of crystal growth, the crystalline phase was changed slightly as the FA ratio of coverage surface area increased, indicating that FA was immobilized on HA by the C-site coordination. From the above, we controlled the FA immobilization states on the HA nanoparticles by the P-site substitution and the C-site coordination through the FA addition timing in the synthesis. Since the -COO^- ions in FA could be selectively substituted with the P-site in HA, it is possible to directly coordinate the foreign organic molecules to the Ca²⁺ ions in HA. Therefore, the immobilization technique of this study is expected to achieve two different drug molecules with diagnosis and therapy functions (i.e., theranostics) on one nanoparticle.

We fabricated three-dimensional (3D)-printed polycaprolactone (PCL) and PCL/graphene oxide (GO) (PGO) scaffolds for bone tissue engineering. An anti-inflammatory and pro-osteogenesis drug dexamethasone (DEX) was adsorbed onto GO and a 3D-printed PGO/DEX (PGOD) scaffold successfully improved drug delivery with a sustained release of DEX from the scaffold up to 1 month. The physicochemical properties of the PCL, PGO, and PGOD scaffolds were characterized by various analytical techniques. The biological response of these scaffolds was studied for adherence, proliferation, and osteogenic differentiation of seeded rabbit adipose-derived stem cells (ASCs) from DNA assays, alkaline phosphatase (ALP) production, calcium quantification, osteogenic gene expression, and immunofluorescence staining of osteogenic marker proteins. The PGOD scaffold was demonstrated to be the best scaffold for maintaining cell viability, cell proliferation, and osteogenic differentiation of ASCs in vitro. In vivo biocompatibility of PGOD was confirmed from subcutaneous implantation in nude mice where ASC-seeded PGOD can form ectopic bones, demonstrated by microcomputed tomography (micro-CT) analysis and immunofluorescence staining. Furthermore, implantation of PGOD/ASCs constructs into critical-sized cranial bone defects in rabbits form tissue-engineered bones at the defect site, observed using micro-CT and histological analysis.